Artificial Organs
25(11):901–906, Blackwell Science, Inc.
© 2001 International Society for Artificial Organs
In vitro Construction of a Potential Skin Substitute through
Direct Human Keratinocyte Plating onto Decellularized
Glycerol-Preserved Allodermis
*Marisa Roma Herson, †Monica Beatriz Mathor, *Silvana Altran, ‡Vera Luiza Capelozzi,
and *Marcus Castro Ferreira
*Plastic Surgery Research Laboratory, University of São Paulo School of Medicine; †Instituto de Pesquisas Energéticas e
Nucleares; and ‡Pathology Division, São Paulo Medical School, São Paulo, Brazil
Abstract: This work demonstrates that glycerol-preserved
acellular allodermis can be used as support for the proliferation of human keratinocytes and that the characteristics
of this bioengineered tissue suggest its possible use as a
permanent skin substitute for therapeutic challenges such
as extensive burns as well as its possible use as an in vitro
model for pharmacological studies. The removal of all
basal membrane components during preparation of the
dermal support also provides an original in vitro situation
that allows observation of the reorganization of the dermal-epidermal junction. The tissue composite obtained is
constituted of dermis covered by a well attached, multistratified epithelium with morphological characteristics
that resemble human epidermis as evidenced by light and
transmission electron microscopy, including the neoformation, albeit incomplete, of the dermal-epidermal junction.
Assessment of involucrin and cytokeratin 14 expression by
immunohistochemical assays established differentiation
patterns. Both immerse and air-liquid interface culture
systems were tested. Key Words: Glycerol conservation—Immunohistochemistry—Keratinocyte culture—
Skin composite—Transmission electron microscopy.
Despite improvement in burn care in the past decades as a result of better understanding of hypovolemic shock following the initial trauma and proper
fluid replacement, establishment of specialized burn
units, the use of topical antimicrobial agents, and the
consensus on the benefits of early surgical eschar
removal, skin replacement remains a challenge. Autologous skin grafting is the gold standard for wound
definitive coverage, but in extensively burned patients, donor areas are scarce. Many alternatives for
temporary cover are already commercially available,
but the search for a biocompatible, permanent, costefficient, off-the-shelf skin substitute continues.
Keratinocyte cultures were described in 1952 (1).
In the 1970s, Rheinwald and Green (2) established a
reproducible method where, from a small skin bi-
opsy, keratinocytes are isolated and cultivated in
vitro to form continuous multilayer epithelial sheets
that can be transplanted as definitive grafts. The dramatic survival of two severely burned children using
this alternative to autologous skin grafting brought
high hopes for burn care specialists (3). Nevertheless, further experience showed that the fragility of
these cultivated epithelia not only required great
technical expertise for cultivating and handling of
the grafts, but also wound bed conditions too scarce
to find in extensive burns in terms of good vascularization and low bacterial counts. The combination of
technical difficulties including time lag to obtain the
needed amount of grafts, high costs, unsatisfactory
graft take, and prolonged instability of the grafts,
has led scientists to search for more resilient substitutes, mostly by the addition of a dermal substitute.
Many alternatives have been proposed, such as
preformed, cross-linked collagen gels (4,5), fibroblast-synthesized extracellular matrix (6), and animal dermis (7). Human allodermis was used as support for autologous keratinocyte cultures in the
Received July 2001.
Address correspondence and reprint requests to Dr. Marisa R.
Herson, University of São Paulo School of Medicine, Av. Dr.
Arnaldo, No. 455, Room 1360, São Paulo 01246-000, Brazil.
E-mail: [email protected]
901
902
M.R. HERSON ET AL.
clinical setup, rendering a permanent wound coverage. First, cryopreserved allogenous skin grafts were
applied to the wound bed; after 5 days, the alloepidermis was abraded, and the vascularized allodermis
was used successfully as support for autologous cultivated keratinocytes (8). The idea of a permanent
skin substitute within this concept was further pursued through in vitro systems where human dermis
was used as support for confluent cultured keratinocyte epithelia (9).
Low-antigenic allodermis can be obtained by decellularization of split thickness skin allografts provided by tissue banks. Although cryopreservation is
the most accepted method of prolonged conservation, skin allografts can also be banked in 85% glycerol (10). The modification of the original work of
Pigossi and colleagues (11) describing the conservation of canine dura mater has proven to be an interesting and less technologically demanding method
for skin preservation. The tissue, although devitalized, retains its morphological structure and can be
used either as a temporary skin substitute or grafted
as a dermal template (12).
This work describes the in vitro construction of a
potentially definitive skin substitute through the direct plating, in high densities, of human keratinocytes onto de-epidermized, glycerol-preserved allodermis, observing the resulting cell morphology and
function and verifying the consequences of exposing
the keratinocytes to the environment using an airliquid culture system (13).
MATERIALS AND METHODS
Keratinocyte cultures
Cell suspensions were obtained from skin fragments devoid of subcutaneous tissue by serial enzymatic cell separation using an 0.05% trypsin/0.02%
EDTA solution (GIBCO-BRL Life Technologies,
Rockville, MD, U.S.A.). The cells were plated at
high density (5 × 106 cells) in 25 cm2 culture flasks.
Cell cultures were fed initially with a mixture of 60%
Dulbecco’s Modified Eagle’s Medium (DMEM;
GIBCO-BRL Life Technologies), 30% Ham F12
(GIBCO-BRL Life Technologies), and 10% fetal
bovine serum (U.S.A. origin) (GIBCO-BRL Life
Technologies), supplemented with 4 mM L glutamine (GIBCO-BRL Life Technologies), 0.18
mM adenine (Sigma Chemical Co., St. Louis, MO,
U.S.A.), 5 ␮g/ml insulin (Sigma), 0.4 ␮g/ml hydrocortisone (Sigma), 0.1 nM cholera toxin (Sigma), 2
nM tri-iodothyronin (Sigma), and 100 IU/ml penicillin/100 ␮g/ml streptomycin antibiotic solution
(GIBCO BRL-Life Technologies). The culture
Artif Organs, Vol. 25, No. 11, 2001
bottles were stored in a 5% CO2 incubator at 37°C.
At the first medium change (i.e., 48 h after the initial
plating), the medium was further supplemented with
10 ng/ml epidermal growth factor (Sigma) and thereafter changed every 48 h. Cell subconfluence was
usually achieved after 5 days. The cells were enzymatically released with 0.05% trypsin/0.02% EDTA
solution (GIBCO-BRL Life Technologies) and
seeded onto preprepared, de-epidermized, glycerolpreserved allodermis.
Preparation of dermis
Fresh split thickness skin grafts obtained from the
Hospital das Clínicas Tissue Bank were placed in
sterile 98% glycerol solution (Central Pharmacy,
Hospital das Clínicas, São Paulo, Brazil). After 24 h,
the tissue was removed from the now diluted initial
solution, the hardened borders trimmed, and then
placed in fresh 98% glycerol solution (11). After 21
days of storage at 4°C, glycerol was removed from
the grafts by bathing the tissue in sterile 0.9% saline
solution (Baxter, São Paulo, Brazil) for 20 min. Epidermal cells were removed by placing precut 1 cm ×
1 cm fragments in a 2.5% Dispase solution (Boehringer-Mannheim Corp., Indianapolis, IN, U.S.A.)
for 45 min at 37°C, followed by a second 15 min bath
in an 0.05% trypsin/0.02% EDTA solution (GIBCOBRL Life Technologies), also at 37°C. The deepidermized dermal fragments, papillary side up,
were adhered individually to multiwell culture plates
by dehydration and then kept moist with DMEM
(GIBCO-BRL Life Technologies).
Immerse and air-liquid culture systems
Cultures were started by plating ∼60,000 cells/cm2
of dermis in culture medium with epidermal growth
factor (EGF) as already described, immersing completely the dermis. The first medium change was performed after 24 h and then every third day for the
next 2 weeks. From the fourteenth to the twenty-first
day of the experiments, culture medium was changed
daily. When an air-liquid interface situation was required, after culturing in the immerse situation for 7
days, the dermal fragments with proliferating keratinocytes on the surface were elevated onto metal
grids, and enough culture medium was added to the
well to keep the dermis in contact with the medium
and the cells exposed to air. But for this modification, cultures were developed in the conditions already described.
Light microscopy
Dermal/keratinocyte fragments (n ⳱ 40) were removed from both the immerse and air-liquid culture
systems for optical microscopy studies at the fourth,
seventh, tenth, fourteenth, and twenty-first day after
IN VITRO-BUILT POTENTIAL SKIN SUBSTITUTE
plating of cells and fixed in buffered 10% formaldehyde solution. Hematoxylin-eosin stained samples
were prepared by standard xylol-alcohol dehydration, paraffin inclusion, and staining. The number of
keratinocytes was established by counting cell nucleus in ten adjoining fields under 1,000× magnification (Labophot, Nikon, Tokyo, Japan) of each specimen. Mean values were calculated and Student’s
t-test applied to evaluate significant statistical differences in cell proliferation.
Transmission electron microscopy
Samples for electron microscopy were obtained
from both culture systems at the twenty-first day after cell plating. After fixation in 2% glutaraldehyde
(Ladd Research Industries, Burlington, VT, U.S.A.)
and postfixation in l% Osmium solution (Ladd Research Industries), the tissue was stained in 0.5%
uranile acetate solution (Electron Microscopy Sciences, Ft. Washington, PA, U.S.A.) and dehydrated
in graded ethanol series (Nuclear, Diadema, Brazil).
It was then embedded in Spurr’s resin (Ladd Research Industries). Thin cuts (75 nm) were placed in
200 mesh grids, covered with 0.25% FormVar (Ladd
Research Industries), and stained with saturated uranile (Ladd Research Industries) and lead uranile
(Ladd Research Industries). The tissue was observed
using a JEM 1010 (Jeol, Tokyo, Japan) transmission
electron microscope, and electron micrographs were
taken with Kodak 4489 electron microscopy film
(Eastman Kodak Co., Rochester, NY, U.S.A.).
Immunohistochemistry
Immunohistochemistry studies for the expression
of cytokeratin 14 and involucrin were carried out in
3 ␮m cuts of the embedded material in 3-aminopropyltriethoxysilane (APTS) (Sigma) after removal of
paraffin with xylol and ethanol (Santa Cruz, São
Paulo, Brazil) and blocking of endogenous peroxidase by 3% H2O2 (Central Pharmacy, Hospital das
Clínicas). Recuperation of the cytokeratin 14 antigen
was carried out by pressure heat (1 min) within a pH
6.0, 10 mM sodium citrate buffer (Synth, São Paulo,
Brazil). Monoclonal antibody to cytokeratin 14 (Novocastra Laboratories Ltd., Newcastle-upon-Tyne,
U.K.) was used in the dilution of 1:40 in PBS (Sigma)
with 1% bovine albumin. Involucrin antigens were
exposed to a 10 min (37°C) enzymatic digestion using a pH 7.0–7.2, 0.25% solution of porcine trypsin
1:250 (Sigma) and reacted against involucrin monoclonal antibodies (Novocastra Laboratories) at a 1:
200 dilution. Following treatment with secondary
biotinylated antibody (Strep-ABC complex, Duet
mouse/rabbit, Dako Corp, Carpinteria, CA U.S.A.)
and 0.6% 3,3⬘-diaminobenzidine (DAB) (Sigma),
903
the final product of the reactions was an intracellular
brownish-gold precipitate. Further contrast was obtained by staining with Harris hematoxylin (20 s).
Positive control reactions were performed in fresh
split thickness human skin.
RESULTS
Exposure of glycerol-preserved split thickness allografts to the enzymatic baths resulted in the removal of epidermal cells and all basement membrane components. Possibly due to culture medium
changes, devitalized dermal cells were washed away
and could not be seen in the histological cuts after 7
days of culture. The seeded keratinocytes adhered to
the dermis and progressed to form a confluent epithelia within 4 days, which progressed to stratification and differentiation. Exposure to air was a strong
stimulus for multiplication and differentiation as
could be well appreciated in hematoxylin-eosin
stained slides at Day 21 of the experiments. In the
immerse culture situation, fewer cell layers could be
seen, the keratinocytes were less differentiated, and
a true corneal layer was absent despite the presence
of some keratin lamellae (Fig. 1). The epithelium in
composites exposed to air showed significantly
higher numbers of cell layers, better cellular organization, and differentiation with the formation of a
true corneal layer (Fig. 2).
Mean cell counts of both immerse and air-liquid
interface cultivated composites epithelia revealed increasing numbers of keratinocytes throughout the
study. Significant statistical differences in cell numbers among composites were evident from the second week on with consistently higher scores in the
air-liquid situation (Table 1).
Transmission electron microscopy allowed for the
observation of good cellular organization in the cul-
FIG. 1. Immerse dermal-epidermal composites are shown at
Day 21 with 4 to 5 cell layers and only primitive corneal layer
(arrow) (light microscopy, ×400).
Artif Organs, Vol. 25, No. 11, 2001
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M.R. HERSON ET AL.
DISCUSSION
FIG. 2. Air-exposed dermal-epidermal composites are shown at
Day 21 with complete epidermal differentiation, as appreciated by
the presence of basal (b), intermediate (i), and superficial (s)
layers as well as a well-formed corneal (c) layer (light microscopy, ×400).
tivated epithelium, including the presence of wellformed intracellular organelles, cellular membranes
with desmosomes, and keratin filaments in more differentiated cells. A true corneal layer was seen in the
composites cultivated in the air-liquid system, but
was absent in the immerse situation where the membranes of cells in contact with the culture medium
presented innumerous microvilli. Functioning melanocytes were confirmed by the presence of intracellular melanosomes. At Day 21 of culture, an incomplete neoformed basement membrane could be
identified in the culture systems by the presence of
well-formed hemidesmosomes, subdense plates, and
adjacent electron dense deposits suggestive of the
reorganization of the lamina densa (Fig. 3).
Immunohistochemistry studies revealed the expression of cytokeratin 14 in basal and suprabasal
layers of the cultivated epithelia in similar locations
as encountered in skin controls (Fig. 4). Involucrin
expression occurred prematurely in all suprabasal
layers of the cultured epithelia, in contrast with
normal skin controls where the antigen was present
only in more superficial and differentiated layers
(Fig. 5).
Following international trends in the search of
skin substitutes, the authors investigated the possibility to build in vitro a skin composite using glycerol-preserved human allodermis as direct support
for the growth of high-density plated human keratinocytes. The choice for human allodermis as support of keratinocyte cultures was based on its natural
and ideal characteristics for growth of these cells.
Allodermis has been shown to be incorporated into
wound beds and has been described as an adequate
support for overgrafting of in vitro cultivated epithelia (14). Preservation of tissues in glycerol is a welldocumented method through which the morphology
of tissues remains intact and which possesses additional advantages over cryopreservation, such as being a technologically less demanding method, and
the fact that tissues can be considered sterile, except
for spores, after 21 days of exposure (15,16). A decrease in tissue antigenicity seems also to occur (17).
In this model, complete removal of basement
membrane components at the time of dermal deepidermization could have represented a hindering
point to cell adherence and proliferation (18). Nevertheless, cells attached, constituted a well-adherent
epithelium to the dermal layer, and an incomplete,
neoformed basement membrane could be observed.
These results render possible the use of this model
for studies of the formation of the basement membrane in vitro where the additional presence of other
cellular (e.g., fibroblasts) or humoral factors can be
controlled.
Cultures were initiated without the presence of a
murine fibroblast feeder-layer as proposed by Rheinwald and Green (2) with the intention to obtain the
least possible immunogeneic skin composite. Some
authors relate late graft loss to antigenic response
originated by remaining xenogeneic fibroblasts (19).
As described previously, plating keratinocytes in
high densities seems to have provided the needed
intercellular stimuli for proliferation (20,21).
TABLE 1. Cell numbers observed in cultivated epithelia throughout the duration of the experiments in immerse (D) and
in air-liquid interface cultures
Experiment
Mean values/p
Day
D1
D2
D3
D4
D5
DI1
DI2
DI3
DI4
DI5
D
DI
p
4
7
10
14
21
1.20
2.75
4.65
5.00
6.20
3.20
5.65
5.30
6.50
5.45
3.70
6.20
6.45
6.90
7.33
2.00
5.40
6.60
7.50
7.98
1.50
0.65
2.60
4.75
5.73
1.20
2.75
5.50
4.00
6.45
3.20
5.65
9.05
8.05
9.55
3.70
6.20
8.65
10.20
9.88
2.00
5.40
6.25
9.35
9.13
1.50
0.65
2.50
9.30
9.68
2.32
4.13
5.12
6.13
6.06
2.32
4.13
6.39
8.18
9.11
*
*
0.172
0.046
0.017
Values represent mean values of counted keratinocyte nuclei (cells) viewed in 10 consecutive “epithelium” fields.
D ⳱ immerse studies; DI ⳱ liquid interface studies; p < 0.05 ⳱ significant.
Artif Organs, Vol. 25, No. 11, 2001
IN VITRO-BUILT POTENTIAL SKIN SUBSTITUTE
905
Striking differences in cell proliferation and cell
maturation could be appreciated due to exposure of
cells to air. Whether the formation of the corneal
layer was stimulated only by the need for a more
efficient cell protection against dehydration or
whether the dermis also played the role of a filter to
culture medium products reaching the cells is yet to
be established.
A certain degree of morphological disorganization
of the epithelium could be appreciated in both culture systems without significant cellular dysplasia.
Hyperproliferative and disorganized epithelia have
been described in conditions of high cell turnover,
such as psoriasis or wound healing, possibly due to
persistence of cell proliferation stimuli beyond
wound closure. Keratinocyte cultures could be considered as a situation where initial cell dispersion
and loss of contact is a strong promoter of the proliferation of epidermal cells with different clonogenic potentials, randomly spread on the culture
plate. Different colony growth potentials result in a
three-dimensional cell confluence irregularity that
can be viewed as cell disorganization in histological
preparations (22). Another contributing factor for
the moderate cell disorganization could be the absence on the dermal support of basal membrane
components such as collagen IV and laminin, accredited with a signaling role for tissue organization (23).
Adequate keratinocyte function and morphology
was documented in transmission electron microscopy by the presence of well-formed junctional
structures such as desmosomes, other intracellular
organelles, and keratin granules in the more superficial layers. Melanocytes are known to persist as
passenger cells in keratinocyte cultures (24). Additional proof of normal function of these cells was the
visualization of pigment granules both in melanosomes and in the cytoplasm of adjacent keratinocytes.
Well-formed hemidesmosomes, subdense plates,
and electron dense deposits are testimony of the
neo-organization of the dermal-epidermal junction.
Complete development of this structure, as well as
normalization of proliferating and differentiating
characteristics, should occur within the presence of
other cells and growth factors. Despite a clear induction for cell proliferation and differentiation toward
the formation of a corneal layer, exposure to air did
not incur improved basement membrane neoformation.
Immunohistochemical studies were considered an
important tool to ascertain the functional characteristics of the cultivated epithelium. Normally, as epi-
FIG. 4. Shown is the expression of cytokeratin 14 in basal layer
of epidermis in normal skin controls (A); premature expression of
the antigen in suprabasal layers in immerse composites at Day
21 of culture (B); similar expression in experiments conducted in
the air-liquid interface at Day 21 (C) (light microscopy, ×400).
FIG. 5. Shown is the expression of involucrin in superficial layers
of normal skin (A); similar locus of expression in immersecultivated composites at Day 21 of culture (B); locus of expression of involucrin in air-liquid-cultivated specimens (C) (light microscopy, ×400).
FIG. 3. Neoformed basement membrane is shown at Day 21 of
culture (immerse situation). Electron-dense deposits resembling
lamina densa (arrows) adjacent to well-formed hemidesmosomes (and subdense plates, H) (transmission electron microscopy, ×80,000, saturated uranile/lead citrate).
Artif Organs, Vol. 25, No. 11, 2001
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M.R. HERSON ET AL.
dermal cells become confluent, stimuli switch from
cell proliferation to basement membrane formation
and cell differentiation, which translates into the expression of corneal envelope constituents, for example, cytokeratin 14 in the basal layer and involucrin in more superficial layers. Thus, the presence of
intracellular keratins can be an indicator that the
expected path toward differentiation has been taken
by the cells. In the proposed dermal-epidermal composite, cytokeratin 14 and involucrin expression occurred in the expected sites. Nevertheless, cytokeratin 14 expression persisted in suprabasal cell layers,
and involucrin expression occurred prematurely.
These differences also occur in hyperproliferative
situations as in psoriasis or cell cultures (25).
Studies in grafting of these composites onto nude
mice have showed promising results with excellent
neovascularization of the dermis, murine fibroblast
repopulation, and survival of the cultured epithelia.
In conclusion, de-epidermized, glycerol-preserved
human dermis is an adequate support for the in vitro
growth of keratinocytes. The resulting tissue composite has morphological and functional characteristics that suggest its potential use as a permanent skin
substitute with benefits from increased mechanical
resistance due to the presence of a dermal component and the in vitro, although incomplete, neoformation of the dermal-epidermal junction.
Acknowledgments: This work was supported in part by
grants from Fundacaõ de Amparo à Pesquisa do Estado de
Saõ Paulo (FAPESP) and from Comissaõ Nacional de Pesquisa Brasil (CNPQ).
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