J. Biomedical Science and Engineering, 2013, 6, 1178-1185 JBiSE
http://dx.doi.org/10.4236/jbise.2013.612147 Published Online December 2013 (http://www.scirp.org/journal/jbise/)
Amniotic membrane as a potent source of stem cells and a
matrix for engineering heart tissue*
Julio Cesar Francisco1,2#, Ricardo Correa Cunha2, Rossana Baggio Simeoni3,
Luiz Cesar Guarita-Souza3, Reginaldo Justino Ferreira1,4#, Ana Carolina Irioda2,
Carolina Maria C. Oliveira Souza2, Garikipati Venkata Naga Srikanth5, Soniya Nityanand5,
Juan Carlos Chachques6, Katherine Athayde Teixeira de Carvalho1,2†
1Bioprocess Engineering and Biotechnology Department, Federal University of Paraná, Curitiba, Brazil
2Cell Therapy and Biotechnology in Regenerative Medicine Research Group, Pelé Pequeno Príncipe Institute, Curitiba, Brazil
3Experimental Laboratory of Institute of Biological and Health Sciences, Pontificial Catholic University of Paraná (PUCPR), Rua
Imaculada Conceição, Curitiba, Brazil
4Federal University of Technology, Curitiba, Brazil
5Department of Hematology, Stem Cell Research Faculty, Sanjay Gandhi Post-Graduate Institute of Medical Sciences, Lucknow,
6Laboratory of Biosurgical Research, Pompidou Hospital, University of Paris Descartes, Paris, France
Email: katherinecarv@gmail.com
Received 28 September 2013; revised 1 November 2013; accepted 19 November 2013
Copyright © 2013 Julio Cesar Francisco et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In accordance of the Creative Commons Attribution License all Copyrights © 2013 are reserved for SCIRP and the owner of the
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Existing therapies for the treatment of chronic heart
failure still have some limitations and there is a
pressing need for the development of new therapeutic
modalities. The amniotic membrane has been used
for the treatment of various diseases, such as con-
junctive defects; however, the mechanisms behind its
repair functions are still unclear. Regenerative medi-
cine is seeking newer alternatives and among them,
biomaterials have emerged in recent years for devel-
oping and manipulating molecules, cells, tissues or
organs grown in laboratories in order to replace hu-
man body part s. Many such ma terials have b een used
for this purpose, either synthetically or biologically,
in order to provide new medical devices. This review
provides a wider view of the regeneration potential of
the use of amniotic membrane as a potential biomate-
rial to facilitate the implementation of new research
in surgical procedures. Amniotic membrane appears
to be an alternative source of stem cells as well as an
excellent biomaterial for cell-based therapeutic ap-
plications in engineering heart tissue.
Keywords: Amniotic Membrane; Heart; Tissue;
Engineering; Stem Cells
The therapeutic options which are currently available for
the treatment of some chronic cardiovascular diseases are
still limited and palliative, highlighting the need for the
development of new therapeutic modalities. Recent ex-
perimental studies have shown great potential, indicating
the possibility of myocardial regeneration by the trans-
plantation of biomaterials, and this has emerged as an
alternative to existing therapies for heart injuries [1].
Amniotic membrane (AM) and amniotic fluid (AF)
have attracted increasing attention in recent years as a
possible reservoir for stem cells and also as promising
sources of stem or progenitor cells that could be useful in
clinical application in regenerative medicine [2,3].
Roubelakis et al. (2012) emphasised that AF and AM
stem cells have the immunophenotypic characteristics of
both adult mesenchymal stem cells and also embryonic
stem cells. Consequently, these cells have been difficult
to identify as they do not have markers and phenotypes.
Roubelakis et al. (2012) proposed the use of a novel ap-
proach to identify them, based on transcriptomic, pro-
teomic, or secretome analyses [4]. Dobreva et al. (2012)
*Potential conflict of Interests: The authors declare that there are no
conflicts of interest.
Sources of funding: This study had no external sources of funding.
#Post-graduate students of the Bioprocess Engineering and Biotechnol-
ogy Department, Federal University of Paraná, Curitiba, Paraná, Brazil.
Corresponding author.
J. C. Francisco et al. / J. Biomedical Science and Engineering 6 (2013) 1178-1185 1179
studied mice, and suggested the use of periostin as a
biomarker of AM [5].
AM has been routinely used in several clinical studies
for the treatment of burns on the skin, ulcers and, pre-
dominantly in ophthalmology, for the treatment of eye-
piece surface disorders. Its use is based on its ability to
improve the process of epithelisation, as well as reducing
the inflammatory processes, angiogenesis and scarring
alopecia [3,6,7].
Tissue-engineered technology provides the possibility
of the enhanced recovery of injured tissues and organs.
In general, this technology involves the selection of an
appropriate substrate cultivation to sustain and promote
the growth of a particular injured tissue. Three-dimen-
sional substrates can be prepared, depending on the tis-
sue in question. Several types of scaffolds are used: syn-
thetic polymers that are not degradable; degradable syn-
thetic polymers; collagen gel, without pores; non-human
collagen gel, with human collagen tissue pores, and de-
cellularised (cadaverous) [8].
In summary, none of the above-mentioned materials is
entirely satisfactory in all respects, i.e. none are immu-
nogenic, antitumoral, resistance and low cost. For this
reason it is necessary to enhance a material for its use as
a substrate for tissue engineering.
The implant composed of a collagen matrix seeded
with cells, has demonstrated benefits in cardiac tissue,
especially when compared to implants using isolated
cells, without matrix. These benefits are extended when
they are combined with other components of the ex-
tracellular matrix, such as growth factors. The amniotic
fluid matrix has advantages because it has a surface area
for cell cultivation with porosity, capillary growth, sta-
bility for mechanical support, biodegradability, and low
immunogenicity [9-13].
Many materials have been used for this purpose,
whether biological or synthetic, with the aim of produc-
ing new medical biomaterials. The purpose of this review
is to provide a broader view of the basic biology of am-
niotic membrane and its potential for use in cardiac re-
Human AM has been used as a biomaterial for surgical
reconstruction for almost 100 years. There has been in-
creasing interest in studying the biology of AM because
it could eventually aid in the treatment of many ailments
and improve the quality of human life. Thus, AM exhib-
its tremendous potential for therapeutic purposes due to
its absorption, high biocompatibility, regenerative capac-
ity and ease of implementation. It is easily accessible and
is without ethical concerns [14].
The use of amniotic membrane originated at the be-
ginning of the twentieth century, when, in 1910, Davis
used it as surgical material for skin transplantation and
subsequently for treating small skin defects in human
patients [15]. In 1940, Roth described the transplantation
of amniotic membrane for the first time in the repair of
siblefaro and conjunctival defects [16]. In 1946, Sorby
and Symons reported good results from the use of AM in
the treatment of acute chemical burns, as well in other
areas of medicine such as in the development of surgical
dressings; the reconstruction of the oral cavity, bladder,
tympanoplasty, arthroplasty and onfalocele; and pre-
venting tissue adhesion in surgery of the head, abdomen,
pelvis, vagina and larynx [6-12,16,17]. The aforemen-
tioned authors introduced the use of AM in ophthalmic
surgery in experimental model and described the ability
of AM to enhance wound healing, and epithelisation,
noting an increase in the use of AM as a biomaterial in
surgeries in recent decades. Similar data were submitted
by other authors over the following years (Table 1) [15,
The fetal membrane is composed of two main layers: the
chorion, which is the outer layer of the placental mem-
Table 1. Amniotic membrane as a potential biomaterial for various organs and tissues. Studies in humans and animals.
Reference Treatment of AM Species Organs/tissues
Davis (1910) [15] Fresh Human Skin
Tseng et al. (1998) [18] Cryopreserved Human Eyes
Azuara-Blanco et al. (1999) [19] Cryopreserved Human Eyes
Chen et al. (2000) [20] Cryopreserved Human Eyes
Mligiliche et al. (2002) [21] Decellularised matrix Rat Peripheral nerve
Tamagawa et al. (2004) [22] Fresh amniotic cells Mouse Hepatocyte
Lo et al. (2007) [23] Fresh decellularised matrix Rabbit Skin
Zao et al. (2005) [24] Amniotic Cells Rat Heart
Tsujhi et al. (2010) [25] Amniotic Cells Rat Heart
Copyright © 2013 SciRes. OPEN ACCESS
J. C. Francisco et al. / J. Biomedical Science and Engineering 6 (2013) 1178-1185
brane which comes into contact with the maternal cells,
and the AM, the innermost layer, which has intimate
contact with the fetus, separated from it only by amniotic
fluid. In the human species, AM appears 7 to 8 days after
conception. Its thickness ranges from 0.02 mm to 0.5
mm and it has not direct blood supply or vascularisation
Human AM, or amnion, is a translucent membrane
composed of a layer of simple epithelium cells firmly
adhered to the innermost layer, called the mesenchymal
layer, which is composed of three layers: compact,
spongy and fibroblastic. These layers contain large
amounts of collagen (type IV and V) laminin and a thick
basal layer as an array, without vascularity. Externally,
the amnion is located in the chorion, comprising a con-
nective tissue that presents fetal vessels (chorioallantoic)
(Figure 1) [19].
Amniotic cells have numerous microvilli on their api-
cal and ventral faces, extending their cellular processes
to the basal membrane, and are known as podocytes.
These cellular junctions are processes of adhesion to the
basal membrane, of the hemidesmosome type with all
their tonofilaments, and are located beneath the basal
membrane, being material that is partially amorphous
and microfibilar [27].
The basement membrane thickness in human tissue
consists of type III, IV, VII collagen, elastin, fibronectin,
perlecan and several other integrin complexes. The base-
ment membrane is known for its healing, neovascularisa-
tion and anti-fibrosis properties [1,28].
Many pinocytic vesicles are found in the cytoplasm of
cells from the AM, with, organelles in abundance, in-
cluding nonlipid reticulum and Golgi apparatus. The cell
cores have an irregular configuration and indents in the
nuclear membrane, with large and homogeneous nucleoli,
suggesting nucleolar activity. The ultra-structure of the
amniotic membrane epithelium has specialised roles,
such as the epithelium lining and as epithelial secretory
with intense intercellular transport and trans-cellular ac-
tivities. AM could be used to reduce inflammation in
scars, enhancing the healing of wounds, and to serve as a
substrate for proliferation and differentiation [29,30].
Amniotic membrane has several biological properties; it
reduces bacterial count and promotes the healing of in-
fected wounds. This membrane is part of an important
group of β-defensin antimicrobial peptides that are ex-
pressed in mucosal surfaces by epithelial cells and leu-
kocytes, which are part of the innate immune system [31].
Its antibacterial property is attributed to its ability to ad-
here to the surface of wounds, to protect injuries and to
reduce pain [32]. The innate immune system has evolved
to eliminate microorganisms from entry in the tissues,
Basement membrane
Fibroblastic layer
Sponge layer
Figure 1. Histological scheme of human amniotic membrane
showing its five layers.
creating antigens that are needed to produce an adaptive
immune response. The absence of leucocytes in the am-
nion allows the use of the halo-transplant procedure,
which does not induce rejection. The amniotic membrane
stroma has several proteases that can inhibit the invasion
of inflammatory cells, which might cause the rapid proc-
ess of apoptosis [6].
Studies have shown the antimicrobial properties of
anmiotic membrane and amniotic fluid in the healing
process. The antimicrobial effect of amnion and chorion
has been demonstrated against large numbers of bacte-
rium, including Streptococcus Group A, Staphylococci
aureus, Escherichia coli and Pseudomonas aeruginosa
Since 1960, there have been many reports demon-
strating biological membranes as an implant material.
Other studies have reported that biological membranes
are organic in nature, inert, and composed almost exclu-
sively of collagen, showing low antigenicity [34].
When amniotic membrane is preserved, it is regarded
as an inert tissue with non-viable cells. The ability of this
membrane to repair tissues occurs through the presence
of growth factors and cytokines [33]. Other studies using
AM have revealed the presence of various growth factors
in the membrane epithelium, such as epidermal growth
factor (EGF); transforming growth factor-β1 (TGF-β1);
keratinocyte growth factor (KGF); fibroblastic growth
factor (β-FGF); and hepatocyte growth factor (HGF),
which act in facilitating cellular migration [35].
Bigbie et al. (1991) demonstrated the role of AM in
the removal of granulation tissue and in promoting the
decrease of exudation in wounds when they were treated
with this membrane. According to Solomon et al. (2001),
AM suppresses the expression of cellular epitopes as
IL-1 α and IL-1 β cytocines. Another unique property of
amniotic membrane is that it does not induce immune
Copyright © 2013 SciRes. OPEN ACCESS
J. C. Francisco et al. / J. Biomedical Science and Engineering 6 (2013) 1178-1185 1181
rejection after transplantation because it does not express
the histocompatibility antigens HLA-A, B, or DR, which
makes it an excellent option for grafting [35-38].
Hao et al. (2000) reported that AM secretes some an-
giogenic factors, such as vascular endothelial growth
factor (VEGF); interleukin-8 (IL-8); angiogenin; inter-
feron-γ; interleukin-6 (IL-6); basic fibroblast growth
factor (bFGF); epidermal growth factor (EGF) and
platelet-derived growth factor (PDGF) [21,22]. It has
also been demonstrated that some anti-angiogenic factors
are released, such as IL-1 receptor antagonist, TIMP3
and TIMP4 [23]. Based on these data, AM can posses
angiogenic and anti-angiogenic properties [38]. Perlecan
induces high affinity binding of fibroblast growth factor
(FGF)-2 to heparansulfate deficient cells or to soluble
FGF receptors; it also possesses angiogenic properties
Regarding the issue of low immunogenicity, clinical
signs of acute infection and rejection were not observed
when amniotic membrane was transplanted in volunteers
[10]. The expression of MHC Class I antigens in AM is
still controversial. Although it has been reported that
HLA-A, B, C and DR was detected in amniotic epithelial
cell culture, the detection of Class 1 antigens in nearly all
cells from the amniotic membrane has been subsequently
reported [10]. In addition, from the observation that am-
niotics cells disappeared without signs of reaction or
rejection, it has been speculated that the short existence
of these cells on the eyepiece surface may be related to
the process of apoptosis [10,11].
There are reports in the literature of the several factors
that are involved in the antifibrotic effect of amniotic
membrane. Fibroblasts are responsible for the process of
healing in wounds enabled by the transforming growth
factor-β (TGF-β); the amniotic membrane has a regula-
tory mechanism of TGF-β and therefore reduces fibrosis
Akle (1981) and Tamagawa (2004) reported that there
was no evidence of tumorigenicity tests when they were
isolated from amniotic membrane cells and transplanted
in human volunteers to analyse immunogenicity [10,22].
Azuara-Blanco et al. (1999) and Dual et al. (2004)
reported that amniotic membrane must be collected from
a healthy donor who shows no signs of any type of infec-
tion, and through caesarean section, where its processing
must be performed under sterile conditions [19,26].
Amniotic membrane contains two different types of
cells: amnion epithelial cells (AECs) and amnion mes-
enchymal stromal cells (AMSCs). Human AECs (hAECs)
are positive for the epithelial markers, cytokeratin 1, 2, 3,
4, 5, 6, 7, 8, 10, 13, 14, 15, 16 and 19 [40]. hAECs ex-
press embryonic stem cell markers, such as OCT-4,
Nanog, Sox-2, Rex-1, SSEA3, SSEA4, TRA-1-60 and
TRA-1-81, and other antigens such as ABCG2/BCRP (a
member of the ATP-binding cassette super family), CD9,
CD24, E-Cadherin, Integrin a6 and b and c-met (receptor
growth factor of the hepatocyte) [41-45].
AMSCs express classical MSC markers (CD90, CD44,
CD73, CD166, CD105 and CD29), as described for bone
marrow stromal cells with the absence of hematopoietic
markers (CD34 and CD45) and the concomitant lack of
monocyte (CD114), macrophage (CD11) and fibroblast
markers. Human AMSCs (hAMSCs) also express low
levels of HLA-ABC but they do not express HLA-DR.
AMSCs are also positive for the pluripotency markers
Oct-4 and Nanog [43-49].
Amniotic membrane has great mechanical strength,
which makes it an attractive biomaterial to be used in
surgery. This membrane is capable of supporting the load
of the pressure of amniotic fluid and repetitive smaller
loads, such as Braxton Hicks contractions during preg-
In most cases, AM is able to withstand loads equal or
close to physiological levels, soon after a transplant, in
order to ensure its resistance. In addition, mechanical
signals can be important mediators of differentiation for
some progenitor cells; AM creates a suitable environ-
ment in the location of new transplanted tissue and it can
increase the strength of the graft, with high levels of ri-
gidity to resist the stress induced during tissue growth
Other biomechanical properties of the amniotic fluid
matrix, such as elasticity, rigidity and viscoelasticity,
depend on the type of proteoglycans, collagen and elastin,
which are important properties, the lack of which can
promote intima layer hyperplasia and occlusion of the
arteries [42].
The cardiogenic differentiation of human amniotic
epithelial cells (hAECs) was first demonstrated by Miki
et al. (2005). The RT-PCR of the cardiac-specific genes,
atrial and ventricular myosin light chain 2 (MLC-2A and
MLC-2V), and the transcription factors GATA-4 and
Nkx 2.5 were expressed in hAECs that were cultured in
media supplemented with ascorbic acid for 14 days. Dif-
ferentiated cardiomyocytes have also expressed α-actinin,
which has been demonstrated by immunocytochemistry
Ilancheran et al. (2007) showed that freshly isolated
native hAECs, and those grown under standard condi-
tions, also expressed the mRNAs of genes that are im-
portant for the specification of the cardiomyoctyic line-
age, such as GATA4, and function, including ANP,
Copyright © 2013 SciRes. OPEN ACCESS
J. C. Francisco et al. / J. Biomedical Science and Engineering 6 (2013) 1178-1185
MYL7, CACNA1C, and KCND3. However, ultrastruc-
tural analysis analysis has revealed that features consis-
tent with relatively mature cardiomyocytes, such as myo-
filaments, myofibrils, H bands and T tubules, were pre-
sent only in differentiated cells [42].
The myogenic differentiation of hAMSCs has been
determined by RT-PCR since Portmann-Lanz et al.
(2006) demonstrated the mRNA expression of myogenic
transcription factors such as myoD and myogenin, and
the protein expression of desmin in hAMSCs after induc-
tion with differentiation media [51].
Fetal maternal stem cell transfer appears to be a criti-
cal mechanism in the maternal response to cardiac injury.
Furthermore, caudal-related homeobox2 (Cdx2) cells
have been identified as a novel cell type for potential use
in cardiovascular regenerative therapy [52].
The use of amniotic membrane as a source for cardiac
regeneration offers excellent advantages in that it is in
plentiful supply and it can be applied immediately, with-
out the need for any cell isolation, which makes it eco-
nomical. In addition, amniotic membrane has good pre-
servability and is immunologically tolerated in alloge-
neic conditions; it has shown cardiogenic differentiation
in vitro and in vivo.
Kim and Tseng, in 1995 and 1998, proposed a method
for the preparation and preservation of amniotic mem-
brane, consisting of collecting the placenta through cae-
sarean section in an environment free from contamina-
tion, removing clots through rinsing with saline solution,
separating the chorion amnion manually, and inserting it
into nitrocelullose with the epithelial surface upwards. In
most studies, the preservation of the membrane follows
the protocol in which the membrane is prepared with
antibiotics and antifungals and kept in medium contain-
ing antibiotics and glycerol at 80˚C [7].
There are reports in the literature about other methods
of preservation and storage of amniotic membrane, in-
cluding freeze-drying, dry air, treatment with glutaral-
dehyde, dispase, gamma irradiation [53]. Dual et al.
(2004) and Mejia et al. (2002) reported that both pre-
served and non-preserved amniotic membrane can be
stored. Dimethyl sulfoxide (DMSO) has been used as an
alternative to the preservation of amniotic membrane by
replacing the rinsing with saline and antibiotics; Azuara-
Blanco et al. (1999) used DMSO to 4, 8 and 10, while
Kubo et al. (2001) used the same product to 0.5, 1.0 and
1.5 M for the washing membrane. After such treatment,
it may be stored in 1.5 M DMSO to 80˚C for several
months [11,19,26,54].
Trehalose is a reductor disaccharide, which is present
in high concentrations in various organisms and is capa-
ble of surviving almost complete dehydration [55]. Na-
kamura et al. (2008) used it to stabilise and preserve cell
membrane proteins. Some studies have demonstrated the
combination of freeze-drying and gamma irradiation of
AM as an efficient sterilisation technique. However, the
use of a chemical family of organic peroxides, peracetic
acid, is usually used as a sterilising agent against many
viruses, bacterium and spores, due to its high oxidation
potential and non-toxic waste generation. In a study by
Wilshaw et al. (2006), the use of peracetic acid to steril-
ise human skin was effective, as it preserved the ex-
tracellular matrix components, such as type IV collagen,
laminin, fibronectin, elastin and glycosaminoglycans.
Furthermore, there was no significant reduction in volt-
age, resistance and elasticity after the treatment of amni-
otic membrane with peracetic acid [56].
Souza et al. (2004) reported the contamination of am-
niotic membrane after caesarean section delivery. These
authors also described the contamination of amniotic
fluid in 13 of 23 patients with intact membranes and sug-
gested aseptic care before handling the membrane. Dual
et al. (2004) argue that there is a risk of infection, and
that disinfecting procedures must be performed, not only
in preparation and storage, but also during clinical use
The use of amniotic membrane associated with tissue
bioengineering is a powerful tool in the repair, protection
and reconstruction of various organs and tissues.
Cargnoni et al. (2009) used a fragment of AM that was
applied to the left ventricle after coronary artery ligation
by ischaemia in rats. Seven days after the transplant, the
rats treated with amniotic membrane showed a greater
preservation of cardiac dimensions and cardiac contrac-
tile element function, and they had improved in terms of
the largest left ventricle ejection fraction shortening and
thickening of the wall. It has been suggested that the use
of amniotic membrane can be the vehicle for the supply
of cells that are able to produce soluble factors and car-
dioprotectors, and this reinforces the notion that this tis-
sue is a source of cells with clinical potential that has not
been fully revealed [58].
A study was performed using transplanted amniotic
membrane cells (cells that are derived from the meso-
dermal lineage). When these cells were transplanted into
rat myocardium in myocardial infarction, the analysis of
heart function demonstrated that 2 and 6 weeks after the
implantation there was no attenuation or improvement of
dilation of the left ventricular dilatation and maintenance
of cardiac dysfunction, when compared to the control
group [59].
Zhao et al. (2005) used isolated cells from amniotic
membrane that were analysed by the PCR technique for
the detection of specific cardiac genes and subsequently
Copyright © 2013 SciRes. OPEN ACCESS
J. C. Francisco et al. / J. Biomedical Science and Engineering 6 (2013) 1178-1185 1183
transplanted them in heart infarction in murine model.
They described that the cells from the amniotic mem-
brane survived in scar tissue for at least two months af-
terwards and these cell were differentiated from cells
with characteristics of cardiomyocytes [24].
Lionetti et al. (2010) reported that transplantation of
human amnion-derived mesenchymal stem cells with
hyaluron in mixed esters of butyric and retinoic acid con-
ditions in rat injured myocardium, differentiated into
cardiomyocyte phenotype and increased the capillary
density of the infarcted myocardium [60].
Tsujhi et al. (2010) studied stem cells derived from rat
heart that were transplanted in AM and were able to be
transdiferenciated in cardiomyocytes. They demonstrated
immunological tolerance in vivo for four weeks after the
transplant. Those experiments were performed without
the use of immunosuppressive agents [29].
Studies suggest that the cells of AM have some com-
mon characteristics with cardiomyocytes, and there is the
possibility of them being suitable for cellular cardio-
myoplasty, although their characteristics and potential
are not yet completely understood [24,49]. In the poten-
tial alternative therapeutic applications in engineering
heart tissue, there should be kinds of disadvantages to be
taken into consideration: allogeneic reactions and the
virus contamination of AM, as Cytomegalovirus [61].
Those conditions can be avoided with the use of immu-
nosuppressant and gamma radiation into End-products:
matrix or cells, respectively.
AM is an alternative source of stem cells, which is par-
ticularly interesting due to its ability to differentiate, low
immunogenicity, low carcinogenicity, as well as being
without any ethical concerns. AM has great potential for
therapeutic applications in regenerating heart tissue and
is a potential source of mesenchymal stem cells, as well
as the source of biocompatible matrix.
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