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Philippine Journal of Science
132 (2): 77-83, December 2003
ISSN 0031 - 7683
77
Radiosensitivity of Angiogenic and Mitogenic
Factors in Human Amniotic Membrane
Custer C. Deocaris1,2,5*, Chester C. Deocaris3, Sonia D. Jacinto4
and Zenaida M. De Guzman5
1National Institute of Advanced Industrial Science and Technology
1-1-1 Higashi, Tsukuba Science City 305-8562, Japan
2Department of Chemistry and Biotechnology, School of Engineering
University of Tokyo, Hongo, Tokyo 113-8656, Japan
3Department of Biology and Biochemistry, UP Manila
4Department of Biology, UP Diliman, Quezon City
5Biomedical Research Section, PNRI, Diliman, Quezon City
*Corresponding author: custer_deocaris@aist.go.jp
+81-29-8613015 fax no.: +81-29-861-3019
The use of human amniotic membranes for
wound coverage was first published 1913 (Sabella,
1913). Until now, outcomes of clinical trials on its
application in supportive care for wound and burn
cases, dermabrasions and skin ulcers, plastic surgery,
laryngology, and for spinal and ocular surgical
procedures are still being reported (Dua & Azuara-
Blanco, 1999). Human amnion is a readily available
biological dressing material that not only prevents
the discharge of plasma from burn wounds, but also
alleviates pain and prevents sepsis. Its is a preferred
wound coverage because the amniotic membrane
stimulates re-epithelization and wound granulation;
reduces loss of fluid, protein, heat and energy;
increases mobility of the injured area; and served as
an ideal wound cover next to the patient’s own skin
(Sharma et al., 1985).
Amniotic membrane as a temporary biological dressing remains as a beneficial and
cost-effective means of treating burns in developing countries. This medical application
is attributed mainly to placental structural and biochemical features that are important
for maintaining proper embryonic development. Since fresh amnions are nevertheless for
straightforward clinical use and for preservation, radiation-sterilization is been performed
to improve the safety of this placental material. However, like any other sterilization
method, gamma-radiation may induce physical and chemical changes that may influence
the biological property of the material. Thus, the aim of this study is to compare the effects
of various levels of radiation-sterilization protocols for human amnions on angiogenic
(neovascularization) and epithelial-mitogenic activities, both of which are physiological
processes fundamental to wound healing.
Water-soluble extract of non-irradiated amnions demonstrates a strong stimulatory
effect on both cell proliferation and angiogenesis. No change in biological activity is
seen in amnions irradiated at 25 kGy, the sterilization dose used by the Philippine Nuclear
Research Institute (PNRI) for the production of radiation-sterilized human amniotic
membranes (RSHAM). However, it appears that amniotic angiogenic factors are more
radiosensitive than its mitogenic components, evident from the depressed vascularization
of the chorioallantoic membrane (CAM) exposed to 35 kGy-irradiated amnions. The dose
of 35 kGy is at present the medical sterilization dose used at the Central Tissue Bank in
Warsaw (Poland) for the preparation of their amnion allografts.
78
In developing countries, the use of human amnions
is cited as the most cost-effective strategy in the
management of burn wounds considering the supply
of the material, efficacy and patient acceptability
(Ramakrishnan & Jayaraman, 1997). Dino et al.
(1966) pioneered the tissue banking of amnions in
the Philippines. However, his group reported practical
difficulties in preserving and applying fresh membranes
in a hospital setting. The first demonstration of the
advantages of radiation processing for the preparation
of human amnion dressings was made possible through
the PNRI-PGH collaboration and the establishment
of a Tissue Bank at the Philippine General Hospital
(Agcaoili et al., 1988). Since then RSHAM has been
manufactured by PNRI for distribution among burn
patients within Metro Manila.
The process of wound healing involves inflammation,
cellular proliferation, tissue remodeling, re-epithelization,
granulation and finally, wound contraction. Two
key factors shape the design and acceptability of
biological wound coverage: the ability to induce cellular
proliferation to promote epithelization-remodeling
at the damaged site and the capacity to recruit new
capillaries by angiogenesis for efficient provision of
oxygen and nutrients to proliferating cells. In fact, the
presence as well as the combination of growth factors
to control appropriate healing is underscored by many
findings. There are five major growth factors that
contribute significantly to the wound healing process:
epidermal growth factor (EGF), transforming growth
factor-b (TGF-b), insulin-like growth factor (IGF),
platelet-derived growth factor (PDGF) and the fibroblast
growth factor (FGF) family. Interestingly, amnion
membrane also possesses a number of important
cytokines, e.g. platelet-activating factor (PAF), basic
FGF, hepatocyte growth factor (HGF), placental
proliferin, TGF-b and EGF (Gospodarwicz, Neufeld
& Schweigerer, 1986; Zhu, Word & Johnston, 1992;
Hansbrough, 1992; Jackson et al., 1994). Autocrine
functioning interleukins (ILs) have been ascribed new
roles in wound healing, i.e. the induction chemotactic
responses of keratinocytes (Michel et al., 1992), the
up-regulation of integrin expression (Reusch et al.,
1990) and stimulation of angiogenesis (Hu, Hori and
Fan, 1993). Amnions also appear to express these
pro-inflammatory cytokines (IL-1, IL-6, IL-8 and tumor
necrosis factor (TNF)-a that activate local protective
response in the presence of invading bacteria as well
as their endotoxins (Trautman et al., 1992; Mitchell et
al., 1991; Dudley et al., 1993). Based on these facts,
several types of biologic dressings and temporary
skin replacements containing many of these growth
factors and cytokines have been developed to enhance
healing yet their applications in human chronic wounds
frequently failed to achieve success. This indicates
that some wounds require a balanced proportion and
an orderly progression of specific growth factors during
healing and such may not be remedied by simple
application of a growth peptide to a synthetic dressing
(Falanga, 1993). Therefore, it is obvious that there is
still a need for continued tissue banking activities of
amnion dressings not only in the Philippines but also
in many parts of the world (Tyszkiewicz et al., 1999,
Gamero & Perez, 1998; Djefall et al., 1998). Despite
the recent developments in tissue biotechnology
wherein new biomaterials are being synthesized, none
could perfectly mimic the function and physiology of a
regenerating skin.
This study was designed to directly analyze the
effects of various levels of absorbed radiation dose used
during the preparation of human amnion dressings. We
examined the differences in the stimulating growth of
epithelial cells in vitro and in recruiting capillaries on
the CAM to understand the fundamental determinants
of wound healing, that is epithelial mitogenicity and
angiogenesis (neovascularization).
Materials and Methods
Sample Collection and Preparation. Amnion
membrane was obtained from 155 mothers sero-
negative for hepatitis B and HIV from the Obstetric
Department of the East Avenue General Hospital
from June 15-30 2001. HIV testing was performed
at the Research Institute for Tropical Medicine using
agglutination tests. Hepatitis B screening was done
at the PNRI using radioimmunoassay (RIA) for Hep
B Antigen.
Tissues with meconium stain were discarded. Each
healthy placenta was washed with tap water to remove
blood. The amnion membrane was excised, washed
with 5% NaH2ClO4 and lyophilized to 15% moisture
level prior to irradiation. Moisture content was monitored
by gravimetric method. The dried membranes were
packaged in polyethylene bags and irradiated at 15,
25 and 35 kGy in a Co-60 source (AECL Gamma
Cell) at the PNRI (AAMI, 1991; ISO, 1993). Water-
soluble fractions of the irradiated amnion membranes
were freeze-dried and stock solutions of the extracts
were prepared in phosphate buffered saline pH 7.2
(PBS). After filter-sterilization through a 0.2 um pore,
the extracts were stored at –70oC until used for cell
proliferation and angiogenesis assays
Microbiological Tests. Five hundred milligram
of the irradiated samples were aseptically transferred
to a thioglycollate broth (Difco) and incubated at 30-
32oC for 14 days. Turbidity in the medium indicates
presence of microbial contaminants. The samples in
the thioglycollate broth were plated on a Plate Count
Agar (Difco) and incubated at 30-32oC for 2 days.
The plate agar was also examined for colony growth.
Determinations were done in duplicates for three
sample lots.
Deocaris et al.
79
XTT-Based Cell Proliferation Assay. T47D
breast carcinoma cells (ATTC, USA) were maintained
in RPMI 1640 medium with 10% heat-inactivated FCS,
1% penicillin-streptomycin and 1% fungizone (Gibco
BRL). Cells were washed with PBS and dispersed in
0.05% trypsin. Cell suspensions were made with RPMI
containing at 5% FCS (sub-optimal supplementation),
1% antibiotics and fungizone and the concentration was
adjusted to 10,000 cells/ml final assay concentration.
Cell cultures were plated into 96-well microtiter plates
(50 ml/well) and were incubated with 50 ml of the amnion
extracts at various concentrations (10, 20 and 30 mg/ml)
in triplicates. After addition of the extracts, 100 ml of
the XTT labeling cocktail (Boehringger Mannheim
GMBh) was added. The optical densities were read at
450 nm after 17 hours. Cells without FCS and amnion
supplementation served as negative controls while
those with additional 5% supplementation served as
positive control.
Duck CAM Assay. Fertilized 7-day old duck Anas
luzonica (Fraser 1839) eggs were treated with 300 ml of
amnion extracts through “windowing” and incubated for
3 days at 37oC in triplicates of two trials. The CAMs were
dissected and photographed. Untreated eggs served
as negative control while those injected with a prepared
tumor extract served as positive control based on the
premise that tumor extracts have angiogenic activities.
Terminal blood vessels were examined under phase-
contrast microscopy using LPO (100x).
Results
Bioburden Status Before and After Irradiation
The microbiological tests of irradiated amnions gave
negative bioburden. It was interesting to note that the
tests given before irradiation (after lyophilization) were
often negative.
In Vitro Cell Proliferation Assay
When added to a confluent monolayer of T47D
breast carcinoma cell line grown under a sup-optimal
level of growth factor-supplementation with 5% FCS,
water-soluble extracts of amnions significantly induced
cell proliferation after assaying for 17 hours with
XTT compared with cultures added with PBS alone
(p<0.001). However, the cell proliferative capacity of
human amnion did not significantly vary with radiation
dose (Fig. 1).
Induction of CAM Neovascularization
Angiogenesis was induced upon addition of the
human amnion extracts within 3 days. The results are
summarized in Fig. 2 and individual representative
photographs of the treatments are shown. It is
observed that amnion induced-angiogenesis based
on the distribution of capillaries under phase-contrast
microscopy is depressed among CAM with amnion
extracts exposed to 35 kGy.
Discussion
During amniotic sac processing (after separation
of chorion) special attention is paid to ensure that the
epithelial side of amnion is placed directly on polyester
net used as a support. After application on the wound,
the epithelial side with the basement membrane
is facing outwards; this will promote migration,
attachment and spreading of the host cells encouraging
epithelialization. The human amnion allografts are
preserved by lyophilization or freeze-drying and
subsequently radiation-sterilized with a dose of 25
kGy. Since the beginning of 1998 over 500 preserved
RSHAM (with a total surface area over 50,000 cm2)
have been prepared at PNRI and distributed to clinics
and hospitals throughout Metro Manila (PNRI-DOST,
2001). While research on clinical application of RSHAM
is being undertaken in various hospitals, we report our
new findings on the basic science behind the wound
healing activity of human amnions and how radiation
processing of this placental material could possibly
affect its biologic activity.
Cutaneous wound healing is a complex biological
process leading to reestablishment of the epidermal
barrier. In small wounds, the clinical significance of
any differences in the rate of epithelization may be
negligible, however, the role of timely wound closure is
very critical in extensive wounds, such as burns (Scott-
Connor et al., 1988), where the epithelization rates
FIGURE 1. Cell Proliferation is Induced by Presence of
Extracts from Irradiated and Non-Irradiated Human Amnions.
Cultured T47D breast carcinoma cells grown in a 5% FCS
and supplemented with 20 mg/ml of water-soluble extracts
from radiation-treated human amnions were assayed for cell
proliferation after 17 hours by an XTT technique. Each bar
represents the mean + SEM.
Optical Density at 450 nm
0 kGy 15 kGy 35 kGy25 kGy
No Amnion
Extract
FIGURE 1. Cell Proliferation is Induced by Presence of
Extracts from Irradiated and Non-Irradiated Human Amnions.
Cultured T47D breast carcinoma cells grown in a 5% FCS
and supplemented with 20 mg/ml of water-soluble extracts
from radiation-treated human amnions were assayed for cell
proliferation after 17 hours by an XTT technique. Each bar
represents the mean + SEM.
Radiosensitivity of Angiogenic and Mitogenic Factors
80
strongly correlate with morbidity and mortality. Motility
and hyperproliferation of keratinocytes are among the
major events during the re-epithelization and as was
mentioned earlier, there are several peptide growth
factors present in the amnion membrane that could
initiate and sustain wound repair until the skin surface
is fully epithelized (Zeigler, Pierce & Herndon, 1997).
The experiment on the XTT-based proliferation assay
using T47D cancer cells demonstrates the general
(cellular) mitogenic potential of amniotic membrane
extracts. Lacking normal human fibroblasts and
keratinocytes in our laboratory, we used instead
T47 human breast carcinoma cells, a transformed
epithelial line since the molecular basis of cell cycle
is universal regardless of cell type, except that during
carcinogenesis the checkpoints or “gatekeepers” are
frequently deregulated (Peter, Bates & Parry, 1996).
From our data, it can be deduced that the biologic
activity of the amniotic mitogenic factors is unaffected
even after radiation sterilization to 35 kGy. A possible
explanation is the fact that there is a vast myriad of
these growth factors with overlapping functions present
in the placental material that any diminution in the cell
proliferative activity accrued from radiation-induced
molecular damages can be masked by redundancy
and bioavailability of these mitogens.
Within a day, as the epithelial cells at the wound
edge, e.g. in burn traumas, begin to proliferate, a basic
synergism with induction of angiogenesis must be
present to support the rapid transport of oxygen and
nutrient to the nascent cells. Otherwise in physiologically
normal conditions, angiogenesis virtually never occurs
in adult tissues, except in the ovary, the endometrium
and the placenta (Norrby, 1997). Angiogenesis is
operative during embryonic development where the
placenta is converted to a highly vascularized organ
capable of mediating the implantation of the embryo
and the efficient exchange of respiratory gases,
nutrients and wastes between the maternal and the fetal
systems (Ramsey, 1982) and by this virtue, it appears
that amnions are a good source of angiogenic agents
that can be applied for supportive care of wounds in
dire need to be vascularized. The placenta secretes
various hormones to induce placental vascularization,
such as bFGF and angiopoietins (Reynolds & Redmer,
2001), and the recently cloned proliferin (PLF) and
the proliferin-related protein (PRP) both belonging to
the prolactin and growth hormone family, respectively
(Jackon et al., 1994). We are not cognizant of previous
studies that investigated individual radiosensitivities
of known angiogenic agents, however based on our
results of the standard CAM assay for angiogenesis, it
is obvious that these amniotic angiogenic factors are
more radiosensitive compared to those molecules for
epithelial cell proliferation. It was proposed earlier that
amniotic (epithelial) mitogenic factors are so numerous
in the placental material that radiation-induced damage
to cell proliferation activity is dampened; conversely,
it can be inferred based on the target theory (Altman,
Gerber & Okada, 1970) that the loss of angiogenic
activity from amnions irradiated at 35 kGy may be
due to fewer amounts and diversity of these placental
angiogenic agents or those factors that can induce
endothelial cell proliferation.
The choice of using 25 kGy as the generally accepted
dose for sterilization of medical products is widely
used to sterilize grafts (Philips, 1994). However the
application of a 40% safety factor common for medical
devices is considered excessive and would endow
undesirable biochemical properties on the biomaterial
(Tallentire, 1986; AAMI, 1991). While the Philippines
utilize 25 kGy as standard dose, other countries, i.e.
FIGURE 2. Loss of Angiogenic-Stimulation by Human Amnion Extracts Upon Irradiation. (A) Vascularity the chorionallantoic
membrane of 10 Day-Old Duck Eggs treated with 3 mg/egg of amnion extracts. Decline in angiogenic-stimulation is observed
with CAM inoculated with 35 kGy-irradiated amnion. (B) A closer inspection of the microvessels under phase-contrast microscopy
show that the capillaries in 35 kGy-irradiated amnions are sparse compared with non-irradiated and 15- and 25 kGy-irradiated
samples. Arrows indicate the capillaries. Normal vasculature of the CAM is shown in the negative control.
35 kGy25 kGy15 kGy0 kGyPBS
A
B
Whole CAM
(Day 10)
Microscopic
Section
Deocaris et al.
81
Poland, uses 35 kGy (Tyszkiewicz et al., 1999) in their
production of amnion allografts. This sterilization dose
is estimated based on the probability of decimating a
given population of contaminating microorganisms in
order to achieve 10-6 sterility assurance level (SAL)
since the graft is expected to come into contact with
compromised tissues (IAEA, 1990). Based on results by
the Polish Tissue Bank, amnions processed at a higher
dose still maintain good take, adhere to wounds and
persist even 3 weeks after grafting (Tyszkiewicz et al.,
1999). While there is an advantage that the probability
of microbial contamination is much less with the delivery
of a higher radiation dose, our bioburden tests indicate
that processed amnions prior to irradiation are already
free from microbes. Other amnion tissue banks have
also observed zero bioburden of freeze-dried amnions
even prior to irradiation (Hai et al., 1992) and this may
be attributed to good processing procedures and the
presence of anti-microbial compounds in the amnion,
such as allantoin (Hansbrough, 1992). From our
experience, radiation processing at 35 kGy would
already be an overkill and it is possible to lower the
irradiation dose below 25 kGy to lessen the processing
cost of the dressing since the starting material is already
“sterile”. In addition, the reported adherence and “good
take” of the amnion biologic dressings are a function
of some structural molecules such dermal matrix
proteins including collagen, tenascin, vitronectin and
glycosaminoglycans as well as the lack of immunologic
response (Hansbrough, 1987). This property was also
investigated using molecular topographic examination
and other physico-chemical analyses on irradiated
membranes (Deocaris et al., 2004).
Finally, another issue that had recently become a
concern is the transmission risks of HIV and Hepatitis
B associated with human amnion tissue banking which
thereby merits the use of higher radiation levels such
as 35 kGy for graft sterilization. Additional precautions
are currently being undertaken, i.e. pre-donor screening
and washing of amnion with 0.05% sodium hypochlorite
solution. These are integrated in the quality assurance
system in tissue banks (Campbell et al., 1994), including
that of PNRI to improve the safety features of RSHAM.
In a tissue banking operation, the accepted levels of
irradiation of RSHAM should consider all the given factors,
namely, sterility requirements, viral transmission risks and
more importantly, the radiosensitivity of amniotic wound
healing properties as was addressed in this study.
Acknowledgements
We are grateful for the assistance of Ms. Mitos
Tolentino and Ms. Luzviminda Ignacio for initial
preparation of the amniotic membranes. The authors
acknowledge the help of Ms. Yvette Marie Chua
and
Dr. Mafel Ysrael for their critical inputs on the
preliminary manuscript. The work was funded by the
PNRI under the Tumor Biology program and by the
DOST-PCASTRD grant for amnion tissue banking.
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Deocaris et al.
