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Tissue-engineered Oral Mucosa: a Review of the Scientific Literature
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Tissue-engineered Oral Mucosa: a Review of the Scientific Literature
K. Moharamzadeh, I.M. Brook, R. Van Noort, A.M. Scutt and M.H. Thornhill
J DENT RES 2007 86: 115
DOI: 10.1177/154405910708600203
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International and American Associations for Dental Research
CRITICAL REVIEWS IN ORAL BIOLOGY & MEDICINE
Tissue-engineered Oral Mucosa:
a Review of the Scientific Literature
K. Moharamzadeh1*, I.M. Brook1,
R. Van Noort1, A.M. Scutt2, and M.H. Thornhill1
1School of Clinical Dentistry, University of Sheffield, Claremont
Crescent, Sheffield, S10 2TA, United Kingdom; and 2Department of
Engineering Materials, University of Sheffield, Broad Lane,
Sheffield, S3 7HQ, United Kingdom; *corresponding author,
[email protected]
J Dent Res 86(2):115-124, 2007
ABSTRACT
Tissue-engineered oral mucosal equivalents have been
developed for clinical applications and also for in vitro
studies of biocompatibility, mucosal irritation, disease, and
other basic oral biology phenomena. This paper reviews
different tissue-engineering strategies used for the
production of human oral mucosal equivalents, their
relative advantages and drawbacks, and their applications.
Techniques used for skin tissue engineering that may
possibly be used for in vitro reconstruction of human oral
mucosa are also discussed.
KEY WORDS: oral mucosa, tissue engineering, scaffold,
keratinocyte.
(I) INTRODUCTION
utologous grafts taken from a different part of the oral cavity—
A
such as free gingival grafts, buccal mucosal grafts, and palatal
grafts—are commonly used approaches for repairing soft-tissue
defects. Since the cells are taken from the same person, the body does
not reject these grafts. However, there are several problems associated
with autologous grafts, including: donor site morbidity, tissue shortage,
and retention of the original characteristics of the donor tissue. To
overcome these problems, in the early 1980s, investigators introduced
cultured epithelial sheets of human skin and oral mucosa, from a small
biopsy, for the treatment of burns (Madden et al., 1986), and intra-oral
grafting (Lauer et al., 1991; Ueda et al., 1991) (Fig. 1). However, these
epithelial sheets, without supporting substructures, are fragile, difficult
to handle, and apt to contract (Clugston et al., 1991; Cooper et al.,
1993). Advances in tissue engineering provide an alternative approach,
since it permits the three-dimensional reconstruction of skin and oral
mucosa, by culture of keratinocytes alone or with fibroblasts on dermal
matrices in vitro (Fig. 2). Satisfactory clinical results have been
reported for intra-oral transplantation of full-thickness engineered oral
mucosa (Lauer and Schimming, 2001; Izumi et al., 2003a). It has also
been shown that the use of a mucosal composite can assist in epithelial
graft adherence and maturation, and minimize wound contraction and
scarring (Cooper et al., 1993). Apart from clinical use, tissueengineered oral mucosa can be used as an in vitro test model for wound
healing, mucotoxicity, and biocompatibility studies. Since tissueengineered in vitro models take the in vivo anatomical structure into
account, they simulate the clinical situation better than do monolayer
cell culture models (Schmalz, 2002). In the last decade, research has
concentrated on the development and characterization of human oral
mucosal equivalents by introducing new dermal scaffolds and epithelial
cell culture methods. In this article, we review the strategies used for
the production of three-dimensional human oral mucosal models and
their applications.
(II) NORMAL ORAL MUCOSA
Received November 29, 2005; Accepted April 27, 2006
Oral mucosa consists of two distinct layers. The surface epithelium
is supported by a fibrous connective tissue layer, the lamina propria.
In many regions of the mouth, the oral mucosa is attached to
underlying structures by a loose connective tissue component, the
submucosa. These three layers are analogous to the epidermal,
dermal, and hypodermal layers of the skin.
The epithelial layer of the oral mucosa is stratified squamous
epithelium, which may be keratinized or non-keratinized, according to
the region of the mouth. The epithelium exhibits four layers of cells:
the basal layer, spinous layer, granular layer, and the superficial layer,
known as the cornified layer in the skin and the keratinized layer in
oral mucosa. Keratinization involves the transformation of viable
keratinocytes in the granular layer into dead surface cells devoid of
organelles and packed with dense masses of cytokeratin filaments. In
non-keratinized oral epithelium, the granular layer is replaced by the
surface layer, the cells of which lack keratohyaline granules. Basal
layer keratinocytes are progenitor cells that undergo terminal
differentiation as they migrate to the surface. In addition to
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Moharamzadeh et al.
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J Dent Res 86(2) 2007
Figure 1. Monolayer culture of human oral keratinocytes on a collagencoated flask.
keratinocytes, oral epithelium contains non-keratinocyte clear
cells: melanocytes, Langerhans cells, and Merkel cells. Adhesion
between epithelial cells is achieved by desmosomes. The basal
layers are attached to the underlying lamina propria through
hemidesmosomes and the basement membrane, which contains
collagen type IV, laminin, and fibronectin.
Cytokeratins are intermediate filaments found in all types
of epithelia, and are the most fundamental markers of epithelial
differentiation. Cytokeratin profile reflects both cell type and
differentiation status in different types and different layers of
epithelia (Table 1).
The lamina propria consists of an abundant network of type
I collagen fibers, and the deeper layers contain collagen type III
fibers and elastic fibers in various amounts, depending upon the
site. Many fibroblasts are present, but only very occasional
macrophages, plasma cells, mast cells, and lymphocytes are
found. The lamina propria also contains vascular components,
which form extensive capillary loops in the papillae between the
epithelial ridges. Lymphatic vessels, nerves, and nerve endings
are also present, as well as the ducts of salivary glands, whose
acini are usually found in the deeper submucosa. Varying
numbers of sebaceous glands are found in the oral cavity, but
are not associated with hair follicles (Atkinson et al., 2000).
Figure 2. Histological sections of (A) normal oral mucosa biopsy, (B)
tissue-engineered skin, and (C) tissue-engineered oral mucosa.
(III) CULTURED EPITHELIAL SHEETS
(A) Monolayer
human keratinocytes in serial culture in vitro, using a feeder
layer composed of irradiated 3T3 mouse fibroblasts and a
In 1975, Rheinwald and Green introduced a method to grow
Table 1. Cytokeratin Expression in Different Layers of Oral Epithelium
Cytokeratin Number
Stratified
squamous
epithelial
cell types
Neutral-Basic (B, II):
Acidic (A, I):
1
10
4
5
Keratinizing suprabasal cells
Non-keratinizing intermediate
and superficial cells
Basal cells
+++
-
-
+
-
+++
-
+++
-
13
7
14
17
19
++
-
-
+
++
+++ +++
-
(++)*
+
* Basal cells in mucosa but not in skin.
Patterns of cytokeratin expression: - no expression, + weak expression, ++ moderate expression, +++ strongly expressed.
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International and American Associations for Dental Research
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8
18
-
-
-
-
-
-
J Dent Res 86(2) 2007
Tissue-engineered Oral Mucosa
specific culture medium called Green's medium. This method is
frequently used for the culture of keratinocytes and production
of single-layer epithelial sheets. Several investigators have
been successful in culturing sheets of oral keratinocytes
without an irradiated feeder layer (Arenholt-Bindslev et al.,
1987; Lauer, 1994). As has been mentioned, these epithelial
sheets are fragile, difficult to handle, and apt to contract.
Monolayer cultures have been extremely helpful to our study of
the basic biology, and responses to stimuli, of both oral and
skin keratinocytes, and many studies have used them. However,
the oral epithelium and epidermis are complex multilayer
structures, with cells undergoing terminal differentiation, and
monolayer cultures may not be a good model of what is
happening in vivo. Thus, the development of a threedimensional multilayer culture system was a major
breakthrough in epithelial biology and tissue engineering.
(B) Multilayer
The culture of keratinocytes on permeable cell culture
membranes at the air/liquid interface facilitated the
construction of multilayer sheets of epithelium, which resemble
native epithelium and show signs of differentiation, such as
basement membrane formation, different cytokeratin
expression, and keratinization if the origin of the keratinocytes
is keratinized mucosa (Rosdy and Clauss, 1990; Rosdy et al.,
1993). A commercially available in vitro model of oral
epithelium, developed by SkinEthic Laboratories (Nice,
France), consists of a three-dimensional, multilayer culture of
the human TR146 keratinocyte cell line on polycarbonate cell
culture inserts. Since the cells are derived from an oral
squamous cell carcinoma cell line, this tissue model does not
fully differentiate, but does form a non-keratinizing oral
epithelium that has been extensively used for biocompatibility
and other studies. SkinEthic's other product, gingival
epithelium, which is produced by air-liquid interface culture of
normal gingival keratinocytes, produces a keratinized stratified
squamous epithelium similar to normal gingival epithelium.
EpiOral TM and EpiGingival TM were developed by MatTek
Corp. (Ashland, MA, USA). These are three-dimensional
reconstructs of human oral (buccal) and gingival epithelium
that form multilayer, stratified non-keratinized and keratinized
oral epithelium, respectively, which exhibit in vivo-like
morphological and growth characteristics. Both tissue
reconstructs express cytokeratin K13 and weakly express
cytokeratin K14. They also produce naturally occurring
antimicrobial peptides, including human beta defensins.
(IV) CONNECTIVE TISSUE
Fibroblasts, the most common cells in the connective tissue,
can be easily isolated and cultured in monolayers by
conventional cell culture techniques. It has been shown that
fibroblasts cultured on three-dimensional porous scaffolds
produce significantly higher levels of extracellular matrix than
do fibroblasts grown in monolayers (Berthod et al., 1993).
Newly synthesized collagen in three-dimensional cultures of
fibroblasts can be characterized by transmission electron
microscopy.
Fibroblasts play an important role in epithelial
morphogenesis, keratinocyte adhesion, and the formation of the
complex dermal-epithelial junction (Saintigny et al., 1993).
The epithelial phenotype and keratin expression are
117
extrinsically influenced by the nature and origin of the
underlying fibroblasts (Okazaki et al., 2003) and the
mesenchymal substrate (Merne and Syrjanen, 2003). It has
been shown that without fibroblasts in the matrix, the
epithelium ceases to proliferate (Fusenig, 1994), while
differentiation continues (Smola et al., 1998). The significance
of fibroblasts has also been shown by an experiment in which
degenerative vacuolization was seen in co-cultures grown in
the absence of fibroblasts. The use of oral buccal and vaginal
fibroblasts led to a non-keratinized epithelium, in contrast to
cultures with skin fibroblasts, which showed slight
parakeratinization (Atula et al., 1997). Thus, fibroblasts may
influence the differentiation potential of the epithelium toward
that found at the site of origin of the fibroblasts.
(V) FULL-THICKNESS ORAL MUCOSA ENGINEERING
An ideal full-thickness engineered oral mucosa that resembles
normal oral mucosa consists of (Fig. 2):
(1) a lamina propria, which are comprised of a threedimensional scaffold infiltrated by fibroblasts producing
extracellular matrix. This structure can be mimicked by the
seeding of oral fibroblasts into a porous biocompatible
scaffold, and long-term culturing in a fibroblast differentiation medium (Berthod et al., 1993; Black et al., 2005).
Possible difficulties of such artificial structures include:
poor fibroblast infiltration, due to the lack of porosity, the
shrinkage of the scaffold if large numbers of fibroblasts are
seeded, and rapid biodegradation of the scaffold.
(2) a continuous basement membrane separating the lamina
propria and the epithelium. The basement membrane can
be characterized by transmission electron microscopy
showing lamina lucida, lamina densa, and anchoring fibers.
Immunostaining for basement membrane antigens—such
as collagen type IV, laminin, fibronectin, integrins, and
bullous pemphigoid antigen—is also a useful
characterization method (Black et al., 2005).
(3) a stratified squamous epithelium on the basement
membrane, including densely packed keratinocytes that
undergo differentiation as they migrate to the surface. This
may be mimicked by the growth in culture of oral
keratinocytes at the air-liquid interface in a chemically
defined medium, which contains keratinocyte growth
factors such as epidermal growth factor (EGF) (Izumi et
al., 1999; Moriyama et al., 2001; Ophof et al., 2002;
Rouabhia and Deslauriers, 2002; Bhargava et al., 2004).
Significant issues that need to be addressed in the growth
of multilayered epithelial constructs on connective tissue
substrates include keratinocyte invasion into the connective
tissue layer and poor differentiation of the epithelium.
To address these problems and optimize the construction of
full-thickness oral mucosa, one must consider many factors.
These include the choice of (A) scaffold, (B) the cell source,
and (C) the culture medium.
(A) Scaffolds
An important element in oral mucosa and skin reconstruction is
the scaffold that supports the cells. Choosing the right scaffold
with ideal biocompatibility, porosity, biostability, and
mechanical properties is a crucial step in tissue engineering.
Scaffolds used in oral mucosa and skin reconstruction fall
into several different categories: (1) naturally derived
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Figure 4. The co-culture system developed by Moriyama et al. for
fabricating composite cultured oral mucosa. Culture was performed at
the air-liquid interface. (A) Keratinocytes; (B) fibroblasts; (C) collagen
sponge and collagen gel; (D) millipore filter; and (E) steel mesh.
being frozen; lyophilization; and ability to be preserved in
glycerol (Heck et al., 1985; Krejci et al., 1991; McKay et al.,
1994; Ghosh et al., 1997). It has been shown that oral mucosal
substitutes composed of oral keratinocytes cultured on skinderived substrates (DED or AlloDerm) show histological and
immunohistochemical characteristics very close to those of
normal oral mucosa (Ophof et al., 2002). However, close
examination of oral mucosal equivalents based on DED reveals
very limited in vitro fibroblast infiltration in this scaffold, as
compared with normal oral mucosa (Fig. 3B).
Figure 3. De-epidermalized dermis (DED). (A) Macroscopic view. (B)
Histological section of oral mucosa reconstructed on DED.
scaffolds, such as acellular dermis and amniotic membrane;
(2) fibroblast-populated skin substitutes; (3) collagen-based
scaffolds; (4) gelatin-based scaffolds; (5) fibrin-based
materials; (6) synthetic scaffolds, such as polymers; and (7)
hybrid scaffolds, which are combinations of natural and
synthetic matrices.
Naturally Derived Scaffolds
Acellular Dermis
Acellular cadaveric dermis (AlloDermTM) was used by Izumi et
al. (1999) as a scaffold for the tissue engineering of oral
mucosa. AlloDerm is an acellular, non-immunogenic cadaveric
human dermis (Rennekampff et al., 1997) that has a polarity by
which one side has a basal lamina suitable for epithelial cells,
and the other side has intact vessel channels suitable for
fibroblast infiltration (Livesey et al., 1995).
De-epidermalized dermis (DED) has been extensively
utilized for the preparation of human epidermal-dermal
composites (Ghosh et al., 1997; Chakrabarty et al., 1999;
Ralston et al., 1999; Lee et al., 2000; Herson et al., 2001), and
also for in vitro reconstruction of human hard palate mucosal
epithelium (Cho et al., 2000) (Fig. 3). De-epithelialized bovine
tongue mucosa has also been used as a substrate for keratinocyte
culture in vitro (Hildebrand et al., 2002). DED is prepared from
split-thickness skin by the removal of the epidermis and dermal
fibroblasts from the dermis. The advantages that have made the
DED a popular scaffold are: good durability and reduced
antigenicity; ability to retain its structural properties, even after
Amniotic Membrane
The possibility of using human amniotic membrane as a
substrate for culturing oral epithelial cells and its suitability for
ocular surface reconstruction in rabbit has been examined
previously (Nakamura et al., 2003). These investigators developed and characterized an engineered oral epithelium with
numerous desmosomes and attached to a basement membrane
with hemidesmosomes; the cells were able to express keratins 3,
4, and 13.
Fibroblast-populated Skin Substitutes
Fibroblast-populated scaffolds include several commercially
available living skin equivalents. DermagraftTM, developed by
Advanced Tissue Sciences Inc. (Coronado, CA, USA), is a
dermal substitute composed of a biodegradable polymer mesh
populated with dermal fibroblasts (Purdue, 1997). Another
product, ApligrafTM (Graft skin), developed by Organogenesis,
Inc. (Canton, MA, USA), is a composite graft composed of
allogenic keratinocytes grown on a fibroblast-populated bovine
collagen gel (Eaglstein et al., 1995; Gentzkow et al., 1996).
Other living skin substitutes include Orcel TM (Ortec
International Inc., New York, NY, USA), PolyactiveTM (HC
Implants, Leiden, The Netherlands), and Hyalograf 3D TM
(Fidia Advanced Biopolymers, Padua, Italy). The fibroblasts
within these scaffolds proliferate and produce extracellular
matrix and growth factors within 2-3 weeks, creating a dermislike matrix (Gentzkow et al., 1996).
Collagen-based Scaffolds
Pure Collagen Scaffolds
In 1996, Masuda and colleagues developed the first in vitro
full-thickness oral mucosal model by seeding cultured normal
gingival keratinocytes on contracted bovine skin collagen gels
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J Dent Res 86(2) 2007
Tissue-engineered Oral Mucosa
(CCG) containing fibroblasts, and co-culturing in a
reconstruction medium at an air-liquid interface for 10 days.
They obtained a well-differentiated mucosal model, which was
histologically similar to native tissues. Moriyama et al. (2001)
modified this method and developed a composite cultured oral
mucosa utilizing an atelopeptide type I collagen sponge matrix
with CCG. Their mucosal model was composed of (1) a lamina
propria in which fibroblasts were embedded in CCG and a
honeycomb structured collagen sponge, and (2) stratified
epithelial cell layers on the surface of the cultured lamina
propria (Fig. 4). The benefit of this model is that the collagen
gel supports fibroblasts, which provides a suitable substrate for
keratinocyte multilayer formation, and prevents epithelial cell
invasion and island formation in the connective tissue layer
(MacCallum and Lillie, 1990). Laminin expression was
detected between the epithelium and lamina propria in
Moriyama's model. However, type IV collagen expression and
hemidesmosome-like structure were not recognizable.
Furthermore, the fibroblasts embedded in the collagen gel
synthesized little extracellular matrix (ECM), compared with
that synthesized on three-dimensional porous scaffolds
(Berthod et al., 1993). Rouabhia and Deslauriers (2002)
produced and characterized an in vitro engineered human oral
mucosa using bovine skin collagen. Their method consisted of
mixing bovine skin collagen with normal human oral
fibroblasts to produce engineered lamina propria, and then
seeding oral epithelial cells on this matrix and growing them at
an air-liquid interface. In their mucosal model, epithelial cells
expressed the proliferation marker Ki-67 as well as
cytokeratins K14, K19, and the differentiation marker
cytokeratin K10. Keratinocytes interacted with fibroblasts by
secreting basement membrane proteins (laminins), and by
expressing integrins (␤1 and ␣2␤1). They also showed that the
engineered oral mucosa was able to secrete interleukins (IL-1␤
and IL-8), tumor necrosis factor alpha (TNF-␣), and different
metalloproteinases, such as gelatinase-A and gelatinase-B. As a
scaffold, collagen matrix is very biocompatible, but it
biodegrades rapidly and has poor mechanical properties. Crosslinking of the collagen-based scaffolds is an effective method
to improve biostability and mechanical properties (Ma et al.,
2003). However, cross-linking of collagen-based tissues
enhances the tendency toward calcification, which is not
desirable in clinical situations (Nimni, 1995).
Compound Collagen Scaffolds
Several compound collagen-based matrices have been
introduced to improve the function of the scaffolds for tissue
engineering. These include: the collagen-chitosan scaffold (Ma
et al., 2003), collagen-elastin membrane (Hafemann et al.,
1999), collagen-glucosaminoglycan (C-GAG) matrix (Ojeh et
al., 2001), and collagen-GAG-chitosan (CGC) scaffolds
(Vaissiere et al., 2000; Black et al., 2005). Chitosan is a
naturally occurring substance that is chemically similar to
cellulose and is derived from chitin, a polysaccharide found in
the exoskeleton of shellfish-like shrimp or crabs. Chitosan
functions as a bridge to increase the cross-linking efficiency of
glutaraldehyde due to the longer chain of amino groups.
Glycosaminoglycans are essential components of the
extracellular matrix, composed of long, non-branched polymers
of repeating disaccharide units, one of which is an amino sugar.
Possible sources of GAGs include shark cartilage, bovine
119
trachea, and porcine cartilage. GAGs such as chondroitin
sulfate and hyaluronic acid are hydrophilic, attracting large
amounts of water and forming hydrated gels, enabling watersoluble molecules to diffuse rapidly (Atkinson et al., 2000).
Fibroblasts grown within a CGC sponge express a significantly
increased collagen synthesis, compared with that of fibroblasts
embedded in a collagen gel and monolayer culture of
fibroblasts (Berthod et al., 1993).
Gelatin-based Scaffolds
Gelatin-based materials such as gelatin-glucan (Lee et al.,
2003), gelatin-hyaluronate (Choi et al., 1999a), and gelatinchitosan-hyaluronic acid (Mao et al., 2003) matrices have been
developed for skin tissue engineering. The denatured form of
collagen, gelatin, is non-antigenic, fibroblast-attractant, and a
macrophage activator, and promotes epithelialization and
granulation tissue formation (Choi et al., 1999a,b, 2001; Hong
et al., 2001). Glucan is antibacterial, antiviral, and
anticoagulant, and promotes wound-healing activity (Douwes,
2005). Hyaluronic acid is added to improve the biological and
mechanical properties of these scaffolds (Mao et al., 2003).
Fibrin-based Scaffolds
Fibrin matrix has been used for the in vitro construction of
human cartilage, skin, and bone (Ruszymah, 2004). BioseedTM,
developed by BioTissue Technologies GmbH (Freiburg,
Germany), is a skin substitute, composed of fibrin sealant with
cultured autologous human keratinocytes. Fibrin glue matrix
gives sufficient adherent stability to the grafted keratinocytes in
an actively proliferating state. Further advantages are ease of
reproducibility and grafting, as well as a reduction in operating
time and costs (Kaiser et al., 1994).
As mentioned above, natural materials possess many
advantages that have made them popular as scaffolds for tissue
engineering. However, these materials also have some
disadvantages. Many of them are isolated from human or
animal tissue, and are not available in large quantities. They
suffer from large batch-to-batch variations, and are typically
expensive. Additionally, these materials exhibit a limited range
of physical properties. These drawbacks have led some
researchers to consider using synthetic materials to fabricate
matrices for use in tissue engineering of skin and oral mucosa.
Synthetic Scaffolds
Polycarbonate-permeable membranes are used in commercially
available partial-thickness epithelial models (SkinEthic and
MatTek tissue models). Successful use of a biodegradable
segmented co-polymer of poly (ethyleneglycolterephthalate)poly (butylene terephthalate) (PEGT/PBT) in skin tissue
engineering has been reported (El-Ghalbzouri et al., 2004).
This synthetic scaffold has good mechanical properties, and
there is no risk of disease transmission. However, incorporation
of fibroblast-populated collagen or fibrin into the pores of the
scaffold is required for better results. Porous polylactic glycolic
acid scaffold has also been used to construct a lining mucosa in
a tissue-engineered prosthetic mucosa for replacement of a
tracheal defect (Kim et al., 2004). A dermal scaffold composed
of knitted poly (lactic-co-glycolic acid) (10:90)-poly (caprolactone) (PLGA-PCL) mesh has shown superior results in
terms of cell distribution and tissue formation, compared with
results from three natural scaffolds, including equine collagen
foam, AlloDerm, and Chitosan (Ng et al., 2004).
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Table 2. Tissue-engineered Oral Mucosa Equivalents
Reference
Cell/Medium
Scaffold
Application
Masuda, 1996
Normal gingival fibroblasts and keratinocytes/DMEM
plus FCS and CaCl2 added KGM*
Contracted collagen gel
Development
Schmalz et al., 1997
Oral keratinocyte and fibroblast co-culture/DMEM plus FCS
Nylon mesh
Biocompatibility testing
Izumi et al., 1999
Oral fibroblasts and keratinocytes/FCS containing
MCDB-153 medium
AlloDerm
Development
Izumi et al., 2000
Oral fibroblasts and keratinocytes/serum-free culture medium
AlloDerm
Development
Schmalz et al., 2000
TR146 cells from buccal SCC/DMEM plus FCS
Polycarbonate filters
Biocompatibility testing
Cho et al., 2000
Palatal epithelial cells/DMEM and Ham's F12 medium plus FCS
DED
Development
Moriyama et al., 2001
Gingival fibroblasts and keratinocytes/serum containing KGM
Contracted collagen gel
and collagen sponge matrix
Development
Navarro et al., 2001
Oral mucosal keratinocytes/DMEM and Ham's
F12 medium plus FCS
Cross-linked collagen-GAG
matrices
Optimization
Rouabhia and Deslauriers, 2002 Oral epithelial cells/DMEM and Ham's F12 medium plus FCS
Bovine skin collagen
Development
Mostefaoui et al., 2002
Oral epithelial cells/DMEM and Ham's F12 medium plus FCS
Collgen matrix
Biocompatibility testing
Ophof et al., 2002
Oral keratinocytes/DMEM and Ham's F12 medium plus FCS
Alloderm, DED, collagen
type-I, collagen-elastin,
and collagen-GAG
Development and comparison
Hildebrand et al., 2002
Normal epithelial keratinocytes/serum-containing
culture medium
De-epithelialized bovine
tongue mucosa
Development and
characterization
Nakamura et al., 2003
Rabbit oral mucosal cells/serum-containing culture medium
Human amniotic membrane
Clinical (ocular reconstruction)
Izumi et al., 2003a,b
Oral fibroblasts and keratinocytes/serum-free culture medium
AlloDerm
Clinical (intra-oral grafting)
Bhargava et al., 2004
Buccal keratinocytes and fibroblasts/Green's medium
DED
Clinical (substitution
urethroplasty)
Andrian et al., 2004
Oral epithelial cells/DMEM and Ham's F12 medium plus FCS
Bovine skin collagen
In vitro (tissue invasion study)
Claveau et al., 2004
Mostefaoui et al., 2004a,b
Tardif et al., 2004
Oral epithelial cells/DMEM and Ham's F12 medium plus FCS
Collgen matrix
In vitro (Candida studies)
Iida et al., 2005
Oral mucosal cells/chemically defined medium
Acellular allogenic dermal matrix Clinical (burn treatment)
* KGM = keratinocyte growth medium, SCC = squamous cell carcinoma.
DMEM = Dulbecco’s modified Eagle medium.
FCS = Fetal calf serum.
SCC = Squamous cell carcinoma.
DED = De-epidermalized dermis.
GAG = Glycosaminoglycans.
Hybrid Scaffolds
A skin substitute based on a semi-synthetic scaffold made of
benzyl ester of hyaluronan (HYAFF and Laserskin) has been
developed. This scaffold has good in vitro and in vivo
biocompatibility and controlled biodegradability (Zacchi et al.,
1998). A hybrid scaffold of poly (lactic-co-glycolic acid)collagen has recently been used for dermal tissue engineering,
but it has shown greater contraction compared with collagenhyaluronic acid foam (Ng et al., 2005).
In a recent study comparing three different types of dermal
scaffolds, a more efficient connective tissue formation was
observed with use of a compression-molded/salt-leached
PEGT/PBT copolymer, in comparison with lyophilized crosslinked collagen, and collagen-PEGT/PBT hybrid scaffolds. It
was also shown that the thickness, porosity, and
interconnecting pore size are important parameters in the
ability of synthetic scaffolds to control connective tissue
formation (Wang et al., 2005).
fibroblasts and keratinocytes. Fibroblasts are usually isolated
from the dermal layer of the skin or by oral mucosal biopsy,
and are used at early passages for tissue engineering, because
the extracellular matrix production by dermal fibroblasts
decreases as the passage number increases (Takeda et al., 1992;
Khorramizadeh et al., 1999). Keratinocytes can be obtained
from different sites of the oral cavity, such as the hard palate
(Cho et al., 2000), gingiva (Yoshizawa et al., 2004), or buccal
mucosa (Bhargava et al., 2004). Normal human keratinocytes
should also be used at very early passages, but immortalized
human keratinocytes, such as HaCaT cells (Boelsma et al.,
1999) or TR146 cells (Schmalz et al., 2000), can be used at
extended passages in the reconstruction of oral mucosal test
models. However, epidermal differentiation of transformed
keratinocytes is not perfect, since the ultimate steps of terminal
differentiation do not occur (Boelsma et al., 1999), and tumorderived cells are abnormal and not suitable for clinical use.
(B) Cell Source
Another important factor that must be considered in oral
mucosa and skin reconstruction is the type and origin of
(C) Culture Medium
The commonly used culture medium for oral mucosa
reconstruction is Dulbecco's modified Eagle medium (DMEM)Ham's F-12 medium (3:1), supplemented with fetal calf serum
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International and American Associations for Dental Research
J Dent Res 86(2) 2007
Tissue-engineered Oral Mucosa
(FCS), glutamine, epidermal growth factor (EGF),
hydrocortisone, adenine, insulin, transferrin, tri-iodothyronine,
fungizone, penicillin, and streptomycin.
In 2000, Izumi et al. developed and characterized a tissueengineered human oral mucosal equivalent using a serum-free
culture method. In that study, they eliminated the use of serum
and irradiated mouse fibroblast feeder layers, to minimize the
exposure of human graft recipients to xenogenetic DNA or
slow viruses that might be present in irradiated mouse 3T3 cells
and serum, respectively. Yoshizawa et al. (2004) used the same
technique to produce human conjunctiva and oral mucosa
equivalents.
It has been demonstrated that perfusion of oral
keratinocytes with medium further enhanced cell viability and
proliferation when cultured in a porous three-dimensional
matrix of collagen-GAG cross-linked with glutaraldehyde
(Navarro et al., 2001).
A summary of different tissue-engineered oral mucosal
models, their cells, culture systems, scaffolds, and their
applications is shown in Table 2.
(VI) APPLICATIONS OF ENGINEERED ORAL MUCOSA
There are generally two major applications for tissueengineered oral mucosa: (1) clinical applications, and (2) as in
vitro test systems and models. It is important to realize that
tissue-engineering approaches may be different for each
purpose. As an example, for clinical applications such as
grafting, transplantation, and guided tissue regeneration, a
biodegradable scaffold with optimal mechanical properties is
desirable, because it will be replaced by the host tissue, and it
must resist natural forces in the oral cavity, while a nonbiodegradable scaffold may result in a foreign body reaction.
Also, transmission of infection and tissue rejection are major
issues. Tumor-derived or virally transformed cell lines are
precluded from clinical use, and high standards of tissue
production and quality control are essential. However, these are
less of a problem for an in vitro test model. Indeed, the need for
reproducibility and lack of batch-to-batch variability may make
the use of tumor and virally transformed cell lines desirable in
this case. The scaffold should have maximum biostability to
maintain its structure throughout the testing procedure.
Depending on the biological endpoint, both epithelium-only
and full-thickness oral mucosal models may have a use for in
vitro tests.
(A) Clinical Applications
Skin Substitutes
Currently, there is a variety of different commercially available
skin substitutes for clinical applications. For example,
DermagraftTM is used for temporary wound coverage of burns,
and is then removed and replaced with autologous skin grafts
(Purdue, 1997). Another product, Apligraf TM , has been
successful in the treatment of venous ulcers (Gentzkow et al.,
1996) and acute wounds (Eaglstein et al., 1995). It has been
reported that tissue-engineered skin can affect the host cells and
promotes tissue regeneration and remodeling by producing
several cytokines and growth factors, such as IL-1, IL-3, IL-6,
IL-8, transforming growth factors ␣ and ␤, and fibroblast
growth factor (Lee, 2000).
Compared with engineered skin, tissue-engineered human
oral mucosa has not yet been commercialized for clinical
121
applications. However, clinical studies have been carried out
with tissue-engineered oral mucosal equivalents for intra- and
extra-oral treatment, with favorable histological and clinical
results.
Intra-oral Applications
Full-thickness engineered human oral mucosa can be used in
periodontal peri-implant reconstruction and maxillofacial
reconstructive surgery. Izumi et al. (2003a) reported a 100%
"take" rate with intra-oral grafting of engineered oral mucosa.
In their study, the engineered grafts showed clinical changes
indicating vascular ingrowth, and had cytologic evidence of the
persistence of grafted cultured keratinocytes on the surface.
The graft enhanced the maturation of the underlying
submucosal layer associated with rapid epithelial coverage. In
vitro labeling of cultured and subsequently grafted gingival
keratinocytes showed that the transplanted keratinocytes
integrated into the newly formed mucosal epithelium (Lauer
and Schimming, 2001). It has also been reported that the
presence of an intact and viable epithelium influences
secondary in vivo remodeling within the connective tissue layer
of transplanted engineered oral mucosa, by synthesis and
release of cytokines, enzymes, and growth factors (Izumi et al.,
2003b).
Extra-oral Applications
In recent years, several tissue-engineered oral mucosa
equivalents have been developed for extra-oral applications.
Bhargava et al. (2004) reported the successful culture of a fullthickness tissue-engineered buccal mucosa, based on DED,
with good mechanical properties for substitution urethroplasty.
Autologous transplantation of cultivated oral epithelium on
human amniotic membrane has been suggested as a feasible
method for ocular surface reconstruction (Nakamura et al.,
2003). Yoshizawa et al. (2004) developed and characterized
human conjunctiva and oral mucosa equivalents, and suggested
their use as graft materials for eyelid reconstruction.
Lately, the application of a tissue-engineered human oral
mucosal equivalent, based on an acellular allogenic dermis, for
the treatment of a burn wound with satisfactory outcome has
been reported (Iida et al., 2005).
Compared with the transplantation of autologous
keratinocytes alone, full-thickness engineered mucosa grafting
results in better and faster wound healing of oral tissues. Longterm clinical follow-up of transplanted engineered oral mucosa
has established this technique as an excellent additional tool in
oral and maxillofacial surgery (Lauer and Schimming, 2002).
However, the commercialization of engineered oral mucosa for
clinical applications is restricted, due to the limitations in
manufacturing facilities, governmental regulatory issues, and
low profitability. Since the best cells for the patients are their
own cells, the use of allogenic cells is not desirable, especially
when most of the indications for many engineered oral mucosal
grafts are not as urgent as the use of skin substitutes in lifethreatening situations, such as extensive burns. Therefore,
tissue-engineered oral mucosa for clinical applications can be
marketed only as a service with limited profitability.
(B) In vitro Applications
In vitro applications of three-dimensional oral mucosal models
include biocompatibility testing and oral biology research
studies, such as disease modeling and wound healing.
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International and American Associations for Dental Research
122
Moharamzadeh et al.
Figure 5. Different biological endpoints for the in vitro assessment of the
response of engineered oral mucosa to an applied stimulus.
Figure 7. Lymphocytes incorporated into tissue-engineered oral
epithelium.
Biocompatibility Testing
Several studies have indicated the use of engineered oral
mucosal models based on collagen membranes and synthetic
polymers as in vitro test models, to evaluate the biological
effects of biomaterials. Mostefaoui et al. (2002), using a
reconstructed human oral mucosal model on a bovine collagen
membrane, examined the effects of dentifrices on tissue
structure and pro-inflammatory mediator released by epithelial
cells. Schmalz et al. studied mucosal irritancy of metals used in
dentistry by introducing these materials onto three-dimensional
fibroblast-keratinocyte co-cultures on nylon mesh (1997), and
also by a 3-D culture of TR146 cells grown on polycarbonate
filters (2000). A similar epithelial model has been used by
several investigators to evaluate the effects of mercury chloride
(Khawaja et al., 2002) and different surfactants (Lundqvist et
al., 2002; Hagi-Pavli et al., 2004) on epithelial viability and
cytokine release from the epithelium. These in vitro models
seem promising for mucotoxicity evaluation of dental
biomaterials, since they reflect the clinical situation better than
do single-layer cell culture test models. Therefore, they can
reduce the need for animal testing and be more specific.
Furthermore, such models allow investigators to study multiple
responses of the epithelium or mucosa to different stimuli (Fig.
5). This is particularly valuable in the testing of responses to
different biomaterials, oral healthcare products, etc., as well as
in studies investigating the response of the oral epithelium or
mucosa to bacteria and other disease processes.
Tissue-engineered Models of Oral Disease
The availability of well-characterized engineered tissue models
J Dent Res 86(2) 2007
Figure 6. Histological picture of Candida albicans colonization and
invasion of tissue-engineered oral epithelium.
has led to the development of a new approach to the study of
disease processes, and the development of tissue-engineered
models of disease. With respect to the oral mucosa, tissueengineered oral mucosal models have been used to assess tissue
invasion by Porphyromonas gingivalis (Andrian et al., 2004) in
a model of gingivitis, and to evaluate the response of the oral
mucosa to Candida albicans infection, such as changes in
basement membrane proteins, matrix metalloproteinase deregulation (Claveau et al., 2004), and inflammatory marker
release (Mostefaoui et al., 2004a,b; Tardif et al., 2004;
Thornhill et al., 2005) (Fig. 6). Further research is in progress
with regard to models of oral mucosal HIV infection and
transmission (Nittayananta et al., 2004), and other disease
models are currently under development.
(VII) FUTURE DEVELOPMENTS
Research on the optimization of oral mucosal equivalents is
still in progress, and scientists are trying to simulate native oral
mucosa as closely as possible, by developing new types of
natural scaffolds, including most of the extracellular matrix
elements, or synthetic scaffolds with optimized porosity and
biodegradability. Other developments to reflect the in vivo
situation more accurately include the incorporation of other
types of cells, including endothelial cells, to promote
angiogenesis and revascularization (Sahota et al., 2004), and
immune cells such as lymphocytes (Fig. 7), monocytes, and
Langerhans cells, to reproduce and evaluate immune responses.
The development of tissue-engineered models of oral disease
will also enhance our understanding of disease processes and
the discovery of new treatments.
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