Burns 27 (2001) 254– 266
www.elsevier.com/locate/burns
Porcine small intestinal submucosa (SIS): a bioscaffold supporting
in vitro primary human epidermal cell differentiation and synthesis
of basement membrane proteins
Kristina Lindberg *, Stephen F. Badylak
Department of Biomedical Engineering, 1296 A.A. Potter Building, Room 204, Purdue Uni6ersity, West Lafayette, IN 47907 -1296, USA
Accepted 21 September 2000
Abstract
The growth pattern of human epidermal cells, fibroblasts or Swiss mouse 3T3/J2 fibroblasts cultured upon the extracellular
matrix (ECM) derived from small intestinal submucosa (SIS) was evaluated. The cell/SIS composites were grown submerged, then
maintained in air/liquid interface for 2, 7, 10 or 14 days. The presence of differentiation-related keratins 10, 14 and 16, FN,
laminin, collagen type VII and collagen type IV was determined by immunohistochemical methods in SIS alone and in the SIS/cell
composite. Only FN could be detected in SIS alone. SIS supported the formation of an epithelial structure with suprabasal
expression of K16 and regional suprabasal expression of K10. The epidermal cells were K14 positive and tended to ‘invade’ the
SIS to various degrees. Following the growth of epidermal cells and fibroblasts on the SIS substratum, immunolabeling of FN,
laminin, collagen type VII and collagen type IV was observed in a cell-associated pattern. The fibroblasts commonly invaded the
SIS, when co-cultivated with epidermal cells on the opposite side of the SIS. The ability of SIS to support epidermal cell/fibroblast
attachment, migration and/or proliferation and differentiation with deposition of basement membrane (BM) components indicates
that the composite model may be useful for studying cell-matrix interactions and for investigation as a dermal substitute. © 2001
Elsevier Science Ltd and ISBI. All rights reserved.
Keywords: Fibroblast attachment; Human epidermal cells; SIS
1. Introduction
Small intestinal submucosa (SIS) is an acellular biomaterial usually derived from the jejunum of pigs [1].
SIS consists predominantly (\90%) of collagens types
I and III similar to the dermal component of skin [2].
Other extracellular matrix (ECM) components of SIS
include small amounts of collagens types VI, V and VI,
but not collagen type VII (unpublished data). The
presence of fibronectin (FN) [3], chondroitin sulfates (A
Abbre6iations: BM, basement membrane; ECM, extracellular matrix; FN, fibronectin; SIS, small intestinal submucosa; STSG, splitthickness skin grafts.
* Corresponding author. Tel.: +1-765-4942994; fax: +1-7654941193.
E-mail address: [email protected] (K. Lindberg).
and B), heparin, heparan sulfate, hyaluronic acid [4],
and cytokines, such as basic fibroblast growth factor
(FGF-2) and transforming growth factor-b (TGFb) [5]
have been documented in the SIS-ECM. These components are known to be important for tissue remodeling
and wound healing, especially in full-thickness skin
defects [6].
The FN content is 0.089 0.05% of dry weight SIS
and is distributed transmurally [3]. This glycoprotein is
found in very small amounts in normal skin, but is
rapidly synthesized by fibroblasts, endothelial cells and
monocytes in soft tissue wound sites, where it acts as a
template for collagen matrix assembly [7]. During granulation tissue formation, FN promotes attachment and
migration of epidermal cells, fibroblasts, endothelial
cells and monocytes. FN also promotes basement membrane (BM) assembly [8]. As wound healing progresses,
the production of FN ceases [9].
0305-4179/01/$20.00 © 2001 Elsevier Science Ltd and ISBI. All rights reserved.
PII: S 0 3 0 5 - 4 1 7 9 ( 0 0 ) 0 0 1 1 3 - 3
K. Lindberg, S.F. Badylak / Burns 27 (2001) 254–266
In animal models SIS has been shown to serve as a
resorbable scaffold for in vivo regeneration/remodeling
of a variety of specialized structures including the urinary bladder [10 –13], dura mater [14,15], anterior cruciate ligament [16], Achilles’ tendon [17], arteries and
veins [18,19] and abdominal wall [20]. Features common to the SIS-induced remodeling process include a
rapid angiogenic response, a mononuclear cell infiltrate
during the early post-implantation period and the deposition of a neomatrix that replaces the SIS scaffold. The
cause of the host tissue response to xenogeneic SIS is
presently unclear, but its three-dimensional structure
and composition appear to represent a favorable substrate for wound healing. SIS-ECM has previously been
shown to support attachment and proliferation of epidermal cells and fibroblasts [21]. During the past 18
months, SIS has been used successfully in over 4000
human patients for the treatment of burns, chronic
non-healing venous stasis and diabetic ulcers, orthopedic soft tissue defects, and as a vaginal sling for the
treatment of urinary incontinence (personal communication with Mark Bleyer, Cook Biotech Inc., and Chris
Butler, DePuy, Inc.). The remodeling process of SISECM implants in humans is comparable with other
mammals.
The presence of dermis or a dermal substitute in
split-thickness grafts plays a critical role in the prevention or minimization of contraction [22 – 24]. Collagenbased materials used as dermal substitutes are favorable
substrates for recellularization and revascularization
and offer promise for the development of next generation dermal substitutes [25 – 27].
Tissue cultivation on plastic is the most commonly
used method to study cells in vitro. The phenotype of
cells grown in monolayer cultures is significantly altered
compared with their normal physiological state. Depending on culture conditions, the cells change their
biosynthesis and morphology to that of a less differentiated cell type. However, the cells maintain their intrinsic genotype and will differentiate, when placed in a
permissive environment, for example, when grafted into
an appropriate in vivo location.
In vitro three-dimensional models based on the use
of cadaver dermis [28,29] or collagen lattices or gels
[30,31] as dermal equivalents, have proven to be of
great value for studying cell-matrix and cell-cell interactions. These studies have contributed to the re-evaluation of the ECM as a passive mechanical support
system to an integral signaling medium that coordinates
not only maintenance of a normal skin structure, but
also the events of wound healing.
The purpose of the present study was to evaluate the
pattern of cell growth, the degree of cell differentiation,
and changes of matrix composition, when epidermal
cells and fibroblasts are grown in vitro upon the SISECM.
255
2. Material and methods
2.1. Preparation of SIS
SIS was prepared as previously described [1]. Briefly,
the superficial mucosal layers and external muscular
layers were removed from the jejunum of pigs within 4
h following euthanasia. The remaining tubular structure consisted of the tunica submucosa and the overlying basilar layers of the tunica mucosa; the most
superficial of which is referred to as the stratum compactum (i.e. the luminal surface). The side of the SIS
material, from which the muscular layers were removed, is referred to as the abluminal surface. The
stratum compactum side of SIS has a smoother surface
than the abluminal side. As shown in Fig. 1a, and b,
the ‘sideness’ of SIS can be easily distinguished by the
use of scanning electron microscopy. The material was
approximately 80 mm thick and was prepared as a
sheet, when the SIS tube was cut in the longitudinal
direction. The SIS was disinfected with 0.1% peracetic
acid and sterilized with 1.5 mRad gamma irradiation,
stored in distilled water at 4°C (hydrated form) or
stored in lyophilized form and re-hydrated in phosphate buffered saline for 20 min prior to use. Unless
indicated otherwise, all experiments were conducted
with the hydrated form of SIS. All experimental conditions described below were tested at least ten times.
The SIS material used in this study was prepared at
the Biomedical Engineering Department, Purdue University, or obtained from Cook Biotech Inc., West
Lafayette, IN.
2.2. Biopsy specimens
The epidermal cells used in this study were derived
from normal skin of a 20-year-old man and from
neonatal foreskin. The human fibroblasts used were
derived from neonatal foreskin.
2.3. Culti6ation of Swiss mouse NIH 3T3 /J2
fibroblasts and human fibroblasts
The NIH 3T3 fibroblasts (a gift from Professor
Howard Green, Harvard Medical School) and the human fibroblasts were grown in Dulbecco’s minimal
essential medium (DMEM) supplemented with 10%
bovine calf serum (Hyclone, Logan, UT).
2.4. Culti6ation of primary human epidermal cells
The epidermal cells were co-cultivated [32] with
lethally irradiated Swiss mouse NIH 3T3/J2 in a 3:1
mixture of DMEM and Ham’s F12 media (Sigma
Chemical Co., St. Louis, MO), supplemented with 10%
fetal calf serum (Hyclone, Logan, UT), 0.4 mg/ml hy-
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K. Lindberg, S.F. Badylak / Burns 27 (2001) 254–266
drocortisone (Sigma Chemical Co., St. Louis, MO), 10
ng/ml human epidermal growth factor (Austral Biologicals, San Ramon, CA), 10 − 10 M cholera toxin (Sigma
Chemical Co., St. Louis, MO), 5 mg/ml Zn-free insulin
(a gift from Lilly Research Laboratories, Indianapolis,
IN), 24 mg/ml adenine (Sigma Chemical Co., St. Louis,
MO) and 2 × 10 − 9 M 3,3%,5 triiodo-L-thyronine (Sigma
Chemical Co., St. Louis, MO).
Fig. 1. Scanning electron microscope picture of the (a) stratum compactum surface, and (b) abluminal surface of SIS. The stratum compactum
surface of SIS is noticeably more smooth compared with the abluminal surface × 500.
K. Lindberg, S.F. Badylak / Burns 27 (2001) 254–266
2.5. Preparation of composites consisting of epidermal
cells and fibroblasts grown on SIS
Swiss mouse NIH 3T3/J2 were seeded at 3 – 4× 105
cells/cm2 on the abluminal surface of SIS. The fibroblasts on SIS were cultivated in DMEM plus 10% CS for
2 – 3 days prior to adding human epidermal cells to the
stratum compactum surface of SIS.
Preconfluent primary epidermal cell cultures in the
third or fourth passage were seeded onto the stratum
compactum surface of SIS at a density of 3 – 4×105 cells
per cm2 SIS. After 3 – 4 days growth in submerged
condition, the composites were lifted to the air/liquid
interface and grown for 2, 7, 10 and 14 days.
2.6. Fibroblasts grown on the stratum compactum
surface or the abluminal surface of SIS with and
without epidermal cells on the opposite surface
Swiss mouse NIH 3T3/J2 fibroblasts or human fibroblasts were seeded at 3 – 4×105 cells/cm2 on the abluminal
or the stratum compactum surface of SIS and cultivated
in DMEM plus 10% CS for 2 – 3 days. Human epidermal
keratinocytes were then seeded at 3 – 4× 105 cells/cm2
SIS on the opposite surface of SIS and grown for 3–4
days submerged in DMEM/F12 medium with supplements. Alternatively, composites containing fibroblasts
only were grown for another 3 – 4 days submerged in
DMEM/F12 medium with supplements. The composites
were then lifted to the air/liquid interface and maintained for 10 days in DMEM/F12 medium with supplements.
2.7. Epidermal cells grown on the stratum compactum
surface of SIS or lyophilized (and rehydrated) SIS
Human epidermal cells were grown on the stratum
compactum surface of SIS as described above using SIS
that had been stored in hydrated form or in lyophilized
form and then re-hydrated prior to use.
2.8. Histological and immunohistochemical e6aluation
of the composites
At the time points indicated above, the composites
were fixed in 10% neutral buffered formalin, paraffin
embedded, sectioned and stained with Hematoxylin and
Eosin (H and E) or frozen and sectioned for immunoperoxidase staining using the Vectastain avidin-biotin-per-
257
oxidase technique (Vector Laboratories, CA, USA) or
standard immuno peroxidase staining techniques. The
tissue sections were evaluated using light microscopy.
The following primary anti-human mouse monoclonal
antibodies were used; Anti-cytokeratin 10 (1:75 dilution), anti-cytokeratin 14 (1:20), anti-cytokeratin 16
(1:40) (all obtained from Novocastra Laboratories, UK),
anti-collagen type VII (1:100) (Chemicon, CA, USA),
LH7.2 (an anti-collagen type VII antibody obtained as
a gift from Irene Leigh, London Hospital) and antilaminin (1:2000) (Sigma, MO, USA). The anti-laminin
antibody and the LH7.2 antibodies cross-reacted with
porcine tissue. Primary polyclonal rabbit anti-human
antibodies to collagen type IV (1:50) (ICN Biomedicals
Inc, Chemical Credential, OH, USA) and to FN (1: 400)
(Sigma, MO, USA) both cross-reacted with porcine
tissue. Secondary antibodies used were peroxidase-conjungated anti-mouse IgG (1:1000) and anti-rabbit IgG
antibody (1:1000) (Sigma, MO, USA). As negative
controls, the tissue sections were reacted with primary or
secondary antibodies alone. Frozen sections of human
foreskin or porcine skin were used as positive controls.
3. Results
3.1. The pattern of cell growth in epidermal
cell/fibroblast SIS composites
By day 2 in air/liquid interface, the epidermal cells
seeded onto SIS constituted a two to three layer thick
epithelial structure. After a total number of 10–11 days
in culture, small epidermal cell clusters were intermittently found beneath the surface of the SIS (Fig. 2a). By
days 10 and 14 in air/liquid interface (after a total of
13–18 days in culture), a several cell layer thick stratified
squamous epithelial structure had formed (Fig. 2b and
c). The ‘invading’ epidermal cells showed a preference
for migrating into existing tube-like structures within the
SIS (Figs. 2c and 9a).
The 3T3 fibroblasts commonly migrated and/or proliferated into the SIS and could be seen transmurally as
well as in close proximity to the keratinocytes (Fig. 2c).
3.2. Fibroblasts grown on the stratum compactum
surface or the abluminal surface of SIS with and
without epidermal cells on the opposite surface
Human and mouse 3T3 fibroblasts seeded on the
abluminal surface of SIS showed the same tendency to
Fig. 2. Epidermal cells derived from a human adult donor and grown on the stratum compactum surface of SIS. (a) At day 7 (Fig. 1a) ( × 400)
in air/liquid interface, clusters of epidermal cells have ‘invaded’ some (arrows), but not all, areas of the SIS matrix. By days 10 (Fig. 1b) ( × 400)
and 14 (Fig. 1c) ( × 200) in air/liquid interface, the epidermal cells constituted a several cell layer thick stratified squamous epithelial-like structure.
Note the lack of keratinocyte migration into the matrix, with exception of one tube-like epithelial structure (arrow head). Swiss mouse 3T3
fibroblasts were seeded onto the opposite surface of SIS 2–3 days prior to seeding the epidermal cells. The fibroblasts can be seen throughout the
matrix (arrows). ×200 H and E staining.
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Fig. 2.
K. Lindberg, S.F. Badylak / Burns 27 (2001) 254–266
259
Fig. 3. Swiss mouse 3T3 fibroblasts grown on the abluminal side of SIS for more than 2 weeks. The cells have not ‘invaded’ the SIS matrix as
seen in composites containing an epidermal cell component (see Fig. 1c). ×200 H and E staining
‘invade’ the substratum as fibroblasts that were seeded
on the stratum compactum surface (data not illustrated). The presence of epidermal cells on the opposite
surface from which the fibroblasts had been seeded,
promoted fibroblast invasion compared with when
fibroblasts were seeded alone (Figs. 2c and 3). Fibroblasts derived from human donors invaded the matrix to
variable extents (Fig. 4). After approximately 2 weeks
in culture, sparse numbers of human fibroblasts were
seen throughout the SIS matrix. The majority of the
human fibroblasts were seen in near proximity of the
cell-seeding surface. In contrary, the Swiss mouse 3T3
fibroblasts tended to invade the SIS transmurally and
were evenly dispersed throughout the matrix.
creased with time in coculture with epidermal cells and
fibroblasts and could be seen in association with the
cells (Fig. 7b). In the cell/SIS composites, laminin,
collagen type IV and collagen type VII staining was
present in conjunction with the epidermal cells along
the BM region of the epithelial structure and around
cell clusters within the matrix (Fig. 8a–c).
All epidermal cells were K14 positive (Fig. 9a). K16
staining was seen suprabasally (Fig. 9b). In some areas
of the composites, the keratinocytes tended to stratify
3.3. Epidermal cells grown on the stratum compactum
surface of lyophilized (and rehydrated) SIS
Epidermal cells seeded on the stratum compactum
surface of SIS in the absence of fibroblasts formed a
less organized and well differentiated epithelium than in
the presence of fibroblasts (human or mouse fibroblasts) (Fig. 5). Epidermal cells seeded on the stratum
compactum surface of lyophilized SIS that had been
re-hydrated did adhere to the substratum (Fig. 6), but
subsequently showed signs of terminal differentiation
and failed to form an epithelial structure.
3.4. Histological and immunohistochemical e6aluation
of the composites
Only FN staining was seen throughout the SIS alone.
The amount of FN staining in the SIS (Fig. 7a) in-
Fig. 4. Neonatal human epidermal cells and fibroblasts grown on
opposite sides of SIS. Note that the human fibroblasts (arrows) have
‘invaded’ the matrix to a lesser extent compared with the Swiss mouse
3T3 cells illustrated in Fig. 1c. × 150 H and E staining.
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Fig. 5. Human epidermal cells grown for 14 days on the stratum compactum surface of SIS. In the absence of fibroblasts the keratinocytes tend
to form a less organized epithelial structure than in the presence of fibroblasts (Fig. 2b and c). × 200 H and E staining.
Fig. 6. Human epidermal cells grown for seven days in air/liquid interface on lyophilized and re-hydrated SIS. The epidermal cells consist of a
two to three cell layers thick epithelium displaying features of terminal differentiation. ×200 H and E staining.
and differentiate into a more mature epithelial structure
with suprabasal expression of K10 (Fig. 9c).
4. Discussion
The present study showed that SIS supports the in
vitro adhesion, migration, growth and differentiation of
primary human epidermal cells and that the SIS matrix
itself is altered by the deposition of new BM proteins
from the cultured cells. SIS has several characteristics
that make the biomaterial suitable for in vitro as well as
in vivo support of epithelial cells.
The presence of FN may play a significant role both
for initial attachment of cells to the SIS and for subsequent cell proliferation. FN synthesis by fibroblasts is
one of the factors contributing to the so-called ‘feeder
layer effect’ (i.e. the ability of fibroblasts to support
epithelial cell attachment and growth in vitro). FNcoating and collagen type IV-coating of the tissue culture vessels is commonly used to enhance in vitro
keratinocyte attachment, migration and proliferation
[33]. Primary epidermal keratinocytes placed in culture
rapidly acquire the ability to secrete FN and exhibit FN
receptors [8,33]. In epidermal cell/fibroblast/SIS composites, the epidermal cell-associated immunolabelling
of FN is presumably a result of de novo synthesis by
the keratinocytes. It is possible that the presence of
TGF-b in SIS contributed to the increased expression
of FN by the keratinocytes [34].
Laminin, which is the most abundant glycoprotein in
the BM, was not present in SIS in quantities detectable
by the immunohistochemical techniques used in this
study. However, as shown in this study (Fig. 8a) and by
others [35], laminin is synthesized by cultured keratinocytes. Adhesive glycoproteins, such as FN and
laminin are known to link ECM components to one
another and to cells. The glycoproteins bind with specific receptors on cell surfaces and on BM components,
such as collagen type IV, which accelerates epithelial
cell attachment [8]. Furthermore, SIS contains FGF-2,
which is known to be angiogenic in vivo and to be
mitogenic for cultured keratinocytes, melanocytes, endothelial cells and fibroblasts [36].
K. Lindberg, S.F. Badylak / Burns 27 (2001) 254–266
261
Fig. 7. FN staining of SIS matrix alone (Fig. 7a). Small amounts of staining are scattered along the stratum compactum surface (arrows) and
throughout the matrix. After 14 days in air/liquid interface, the FN staining of the SIS/cell composite (Fig. 7b) is seen in a cell– associated pattern
(arrow head). ×200 immunoperoxidase staining.
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Fig. 8. Immunoperoxidase staining of SIS/cell composites at days 10 – 14 in air/liquid interface using antibodies to laminin (Fig. 8a), collagen type
IV (Fig. 8b) and to collagen type VII (Fig. 8c) showed that these BM products were expressed in a cell-associated pattern (arrows) × 400.
Considering the composition of SIS, the biomaterials’
in vitro cell growth promoting properties are not surprising. Interestingly, the lyophilized (and re-hydrated)
form of SIS did not support growth and differentiation
of the epidermal cells as well as the SIS that had not
been lyophilized. It is possible that the three-dimensional structure and/or composition of SIS had been
altered by the dehydration process, which in turn may
have changed the cell adhesion properties of the matrix.
It is conceivable that modifications of the dehydration
K. Lindberg, S.F. Badylak / Burns 27 (2001) 254–266
263
Fig. 8. (Continued)
Fig. 9. Immunoperoxidase staining of SIS/cell composites using antibodies to keratin 14 (Fig. 9a) labeled all keratinocytes. Note that in one area,
the keratinocytes have migrated into a tube-like structure (arrow). Antibodies to keratin 16 showed a suprabasal staining pattern (Fig. 9b). AntiK10 staining had a regional suprabasal distribution (Fig. 9c) × 200.
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K. Lindberg, S.F. Badylak / Burns 27 (2001) 254–266
Fig. 9. (Continued)
process, such as amorphous-phase freeze drying [37],
would improve the materials in vitro cell supportive
properties.
In addition to the favorable composition of the hydrated SIS/ECM itself for cell growth, the pattern of
cell growth appeared to be modulated by interactions
between the different cell types that were seeded in the
composite. The fibroblasts did not tend to invade the
SIS, when grown without epidermal cells (Fig. 3). Although the normal human fibroblasts did not invade
the matrix to the extent that the immortal 3T3 cells did
(when co-cultivated with epidermal cells), the two cell
types’ ability to support the formation of an epitheliallike structure was comparable. The lesser organization
and differentiation of epidermal cells grown on SIS
without fibroblasts (Fig. 5), compared with when the
K. Lindberg, S.F. Badylak / Burns 27 (2001) 254–266
epidermal cells were grown in the presence of fibroblasts (Fig. 2b and c), was another implication of the
impact of cell-matrix interaction in the SIS composite.
There is accumulating evidence that interactions between epithelial cells and the underlying tissue play a
key role for regeneration and maintenance of site-specific tissue characteristics [38 – 41]. Clinical studies have
shown that the epidermal component of a skin graft is
essential for minimization of morbidity and mortality.
In addition, a suitable dermal substitute can induce the
synthesis of normal dermal components, such as elastic
fibers, which provide grafts with mechanical properties
that mimic the properties of normal skin [42].
The clinical importance of ECM components for skin
wound closure and remodeling is well documented.
Dermal ECM (usually derived from cryo- or glycerol
preserved cadaver skin) significantly improves clinical
results, when used in conjunction with autologous splitthickness skin grafts (STSG) or cultured epidermal
autografts (CEA) for permanent repair of full thickness
skin defects [41]. Evidence for the positive impact of
this ECM/epidermal interaction include the accelerated
regeneration of BM components, such as anchoring
fibrils (collagen type VII), and structural characteristics
of normal skin, such as rete ridges [43 – 46]. Numerous
clinical and animal studies have evaluated the ability of
synthetic and biological substrata/dermal analogs to
support keratinocyte proliferation for constructing a
dermal/epidermal composite. Ideally, this type of construct should provide an ECM scaffold that rapidly
incorporates into the wound bed, promotes connective
tissue remodeling and delivers epidermal cells to the
wound site. The keratinocytes that re-epithelialize a
wound produce a large number of cytokines and ECM
molecules, which are thought to contribute to the accelerated healing reported in different types of ulcers
treated with cultured epidermal keratinocytes [47 –49].
Based on the results in this study, and the reports of
rapid integration and revascularization of SIS, when
the ECM is transplanted to various body sites in animal
models and in humans ([10 – 19] and personal communication with Mark Bleyer, Cook Biotech Inc., and Chris
Butler, DePuy, Inc.), the cell/SIS composite may be
useful as a carrier of epithelial cells to a wound site. It
remains to be determined whether the wound healing
properties of composites with recently seeded epidermal
cells will be superior to composites containing a more
mature epidermal structure. The results of the present
study, combined with the results of previous studies
that evaluated the utility of collagen-based dermal substitutes, suggest that this class of biomaterials may find
utility as either a carrier for keratinocytes or as an
alternative treatment modality for patients with partialthickness and full-thickness wounds [22 – 24].
In the present study, culture conditions allowed de
novo synthesis of BM proteins commonly expressed in
265
monolayer cultures and in three-dimensional in vitro
models (laminin and collagen type IV), and the synthesis of the major protein in anchoring fibrils of the BM,
collagen type VII.
The cell/SIS composite used in these experiments
provides a promising model for studies that could
contribute to understanding the mechanisms that regulate cell adhesion, migration and proliferation of primary human epithelial cells and for studying the
interaction between ECM components, epithelial cells
and mesenchymal derived cells.
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