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ORIGINAL RESEARCH REPORT
Biological characterization of dehydrated amniotic membrane
allograft: Mechanisms of action and implications for
wound care
Marc C. Moore
1
| Paul P. Bonvallet
2
| Sita M. Damaraju
2
| Heli N. Modi
2
|
Ankur Gandhi
2
| Peter S. McFetridge
3,4
1
Stephenson School of Biomedical
Engineering, University of Oklahoma, Norman,
Oklahoma
2
Product Development, Integra LifeSciences,
Princeton, New Jersey, 08540
3
J. Crayton Pruitt Family Department of
Biomedical Engineering , University of Florida,
Gainesville, Florida
4
Department of Molecular Genetics and
Microbiology, University of Florida,
Gainesville, Florida
Correspondence
Peter McFetridge, J. Crayton Pruitt Family
Department of Biomedical Engineering,
University of Florida, Gainesville, FL, USA.
Email: pmcfetridge@bme.ufl.edu
Funding information
Integra LifeSciences, Grant/Award Number: -
Abstract
There is a growing clinical demand in the wound care market to treat chronic wounds
such as diabetic foot ulcers. Advanced cell and tissue-based products (CTPs) are
often used to address challenging chronic wounds where healing has stalled. These
products contain active biologics such as growth factors and cytokines as well as
structural components that support and stimulate cell growth and assist in tissue
regeneration. This study addresses the in vitro biologic effects of a clinically available
dehydrated amniotic membrane allograft (DAMA). The broad mechanism of action
results from DAMA's biologic composition that leads to stimulation of cell migration
cell proliferation, and reduction of pro-inflammatory cytokines. Results show that
DAMA possesses growth factors and cytokines such as EGF, FGF, PDGFs, VEGF,
TGF-β, IL-8, and TIMPs 1 and 2. Furthermore, in vitro experiments demonstrate that
DAMA stimulates cell proliferation, cell migration, secretion of collagen type I, and
the reduction of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. This study find-
ings are consistent with the clinical benefits previously published for DAMA and
other CTPs in chronic wounds suggesting that the introduction of DAMA to non-
healing, complex wounds helps to improve the wound milieu by providing essential
structural components, cytokines, and growth factors to create an appropriate envi-
ronment for wound healing.
KEYWORDS
cytokines, growth factors, wound healing
1|INTRODUCTION
Wound healing is a complex process that is characterized by three dis-
tinct phases: inflammation, proliferation, and remodeling (Broughton
et al., 2006; Enoch & Leaper, 2005; Falanga, 2005; Kirsner &
Eaglstein, 1993; Velnar et al., 2009). During the initial stage of wound
healing, platelets flood the wound site to begin the clotting process as
well as release multiple chemical signals to begin a cellular signaling
cascade. In this phase, the inflammatory response results in phagocy-
tosis of debris and dead or injured cells. During the phase transition
from inflammation to proliferation, growth factors and cytokines such
as epidermal growth factor (EGF), transforming growth factor beta
(TGF-β), and platelet-derived growth factor (PDGF) are released
(Barrientos et al., 2008; Stojanovic et al., 1996; Werner &
Grose, 2017). These molecules signal cellular migration, recruitment
and proliferation, processes that are essential for effective tissue rem-
odeling, vascularization, and epithelialization (Ellis et al., 2018). In
addition, extracellular matrix (ECM) components, including structural
proteins, glycoproteins, and glycosaminoglycans (GAGs), play a critical
role in maintaining the physiological microenvironment and contribute
Received: 28 October 2019 Revised: 31 March 2020 Accepted: 29 April 2020
DOI: 10.1002/jbm.b.34635
3076 © 2020 Wiley Periodicals, Inc. J Biomed Mater Res. 2020;108B:3076–3083.wileyonlinelibrary.com/journal/jbmb
building blocks for the healing process (Cornwell et al., 2009; Schultz
et al., 2011). During the proliferative phase, new ECM forms as cells
respond to signals from fibroblasts, myofibroblasts, keratinocytes, and
inflammatory cells which leads to angiogenesis, extracellular matrix
deposition, and finally re-epithelialization (Hutton et al., 2009; Koob
et al., 2014a; Olczyk et al., 2014; Sheikh et al., 2014; Tseng
et al., 1999). Once re-epithelialization is complete, the remodeling
phase begins where the disorganized ECM proteins such as collagen
are remodeled and organized allowing for an increase in strength.
During the wound healing process, cells and ECM interact in a
bidirectional manner known as dynamic reciprocity, where cells syn-
thesize and reconstruct the ECM and in turn the ECM provides a
favorable environment for cell attachment and proliferation
(McQuilling et al., 2017; Schultz et al., 2011). This interaction is
heavily regulated by cytokines and growth factors. In normal wound
healing, the transition between phases is orderly and the signaling
molecules regulate this process; however, in chronic wounds, progres-
sion stalls at the inflammatory phase (Ellis et al., 2018). During this
stalled state, there is an elevated inflammatory response along with
sustained levels of proteases such as matrix metalloproteinases
(MMPs) which have been shown to regulate the growth factor and
cytokine response (Zhang, 2010) as well as degrade existing and new
ECM, further damaging tissue and preventing the wound from pro-
gressing to the proliferative stage (Schultz et al., 2011; Cutting &
Tong, 2003). In these cases, the clinician must intervene with more
advanced wound care solutions. Studies have demonstrated a clinical
benefit from interventions such as cell and tissue-based products
(CTPs), which incorporate both ECM and growth factors to stimulate
the healing process (Davis, 1910; Fetterolf & Snyder, 2012; Regulski
et al., 2013). In one such study, Snyder et al. (2016) demonstrated that
when patients are treated with either the standard of care or a
dehydrated amniotic membrane allograft (DAMA) in addition to stan-
dard of care that a higher incidence of wound closure occurs in
patients treated with DAMA.
Available CTPs range from decellularized collagen-based sub-
strates to those derived by harvesting tissue from donor humans (allo-
graft/autograft) or animals (xenograft) (Cornwell et al., 2009; Jones
et al., 2002). Allografts of interest are derived from human placental
tissue because of their relative ease of sourcing and composition that
includes a range of functional biological molecules that aid in wound
healing and host-tissue integration (Akle et al., 1981; Bailo
et al., 2004; Erickson & Couchman, 2000; Koob et al., 2013). These
attributes have made these membranes a highly valued tissue source
for use in wound management. Furthermore, these placental derived
membranes have shown favorable biocompatibility due to their low
immunogenicity (Ueta et al., 2002), anti-inflammatory functions (Hao
et al., 2000), and minimal processing. Furthermore, these tissues are
harvested without the ethical concerns associated with human embry-
onic tissues and use of animal sources (Chen & Tofe, 2010; Liu
et al., 2010; Toda et al., 2007). Human placental membranes provide
an abundance of useful components including membrane constitu-
ents, ECM components such as collagen (Parolini et al., 2012), and
growth factors and cytokines which are all critical to support and
stimulate cell growth. When used in chronic wounds such as diabetic
foot ulcers, membranes such as dehydrated amniotic allografts have
been shown to have a positive stimulatory effect on wounds stalled in
the inflammatory phase (Abdo, 2016; Barr, 2014; Lintzeris
et al., 2015; Rosenblum, 2016; Snyder et al., 2016). They contain key
interleukins (ILs), IL-4, 6, 8, and 10, which regulate cell function, act as
chemokines, and stimulate cell proliferation (Kindt et al., 2006). IL-10
further serves an anti-inflammatory role by down-regulating MHC-II
antigens and counteracting other inflammatory factors in the process
of tissue repair (Mosser & Zhang, 2008; Weber & Iacono, 1996).
The objective of this study was to characterize the in vitro cellular
response underlying the observed clinical effects of DAMA in chronic
wounds. Specifically, biochemical and biophysical analyses were per-
formed to verify the retention of structural and biochemical compo-
nents of amniotic membranes post-processing, including the
quantification and identification of growth factors and cytokines. Fur-
thermore, the modulatory effect of these membranes on cell migra-
tion, proliferation, ECM deposition, and inflammation was evaluated
in vitro using human adult dermal fibroblasts.
2|MATERIALS AND METHODS
2.1 |Dehydrated amniotic membrane allograft
DAMA (AmnioExcel, Integra LifeSciences, Princeton, NJ) single layer
amniotic membranes were donated from Integra LifeSciences for
characterization studies. Placental tissues were donated by consenting
women and retrieved at the time of planned Cesarean section during
childbirth. In accordance with the US Food and Drug Administration
(FDA) and the American Association of Tissue Banks (AATB) guide-
lines, placental tissue donors were screened for communicable dis-
eases before processing. DAMA processing and dehydration steps
were completed within a few hours to maintain the tissues distinct
biochemical composition and handling properties.
2.2 |ELISA array
Growth factor content and concentrations were quantified for DAMA
membranes using enzyme-linked immunosorbent assay (ELISA,
RayBiotech Inc.). Membrane samples were weighed, homogenized in
lysis buffer containing protease inhibitors for 24 h at 4C, homoge-
nized again and then centrifuged to remove residual tissue fragments.
Supernatant extracts were used as samples in the ELISA kit assays.
2.3 |Histology
DAMA membranes were rehydrated in 1×PBS for 1 h. The samples
were subsequently fixed in formalin for 24 h, paraffin embedded, sec-
tioned, and stained with Hematoxylin and Eosin (H&E). Imaging was
performed on a Leica Confocal microscope at magnifications of 20×.
MOORE ET AL.3077
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Collagen type I, collagen type III, fibronectin, and laminin were all sta-
ined by immunohistochemistry (IHC) using formalin fixed paraffin
embedded sections. Briefly, slides were dried overnight, baked at
60C for 1 h, deparaffinized in xylene, rinsed in alcohol, rehydrated in
water, and equilibrated in wash buffer (TRIS buffered saline with
0.05% Tween 20; Dako, K8007). Collagen type I stain was carried out
using a rabbit polyclonal antibody directed against Collagen type I
from human placenta (Bio-Rad, 2,150–0020). Collagen type III stain
was carried out using a rabbit polyclonal antibody directed against
Collagen type III from human placenta (Bio-Rad, 2,150–0100). Fibro-
nectin stain was carried out using a rabbit polyclonal antibody raised
against Fibronectin isolated human plasma (Abcam, ab6584). Laminin
stain was carried out using a rabbit polyclonal antibody isolated from
the rat yolk sac tumor cell line L2 (Agilent, Z0097). Next a serum free
protein block was applied for 5 min (Dako, X0909) followed by anti-
Rabbit labelled Polymer-HRP (Dako, K4003) for 30 min and DAB+
Chromogen solution (Dako, K4011) for 5 min. Tissues were counter-
stained in a modified Harris hematoxylin (Dako, S3301) for 5 min.
Also, for collagen type I, collagen type III, laminin, and fibronectin,
human skin tissue was stained (not shown) to validate the specificity
of the antibodies and staining protocols.
2.4 |Extraction protocol
Briefly, 5 cm
2
of DAMA was homogenized (Precellys, Bertin Instru-
ments) in 1 ml of Fibroblast basal media (ATCC) for 4 min. This solu-
tion was then incubated at 37C for 72 h shaking at 300 rpm. Extract
was centrifuged and sterile filtered using a 0.22 μm PVDF filter
(Millipore). In these experiments, 5 cm
2
of DAMA membrane in 1 ml
of media correlates to 100% extract.
2.5 |Cell culture and ECM stain
Passage 4 human adult dermal fibroblasts (hDF, ATCC) were seeded at
5×10
3
cellsperwellin24-wellplatesandculturedin(a)assaymedia
with 1% fetal bovine serum (FBS) for the negative (−) control or (b) 15%
DAMA extract in assay media for 7 days or (c) complete growth media
with 10% FBS as a positive (+) control. Samples were fixed in 4% parafor-
maldehyde for 20 min, nuclei were stained blue with Hoescht 33342
(Thermo Fisher Scientific), and collagen type I was stained with a mono-
clonal rabbit collagen type I antibody (Invitrogen PA1-26204) and Alexa
Fluor 488 goat anti-rabbit secondary antibody (Thermo Fisher Scientific).
2.6 |Cellular migration assays
2.6.1 |Boyden chamber assay
Passage 5 hDF were seeded at a density of 2 ×10
4
cells per well in the
top insert of a 24-well Transwell plate (pore size 8.0 μm, Corning
Costar) and were serum starved overnight. The bottom wells contained
basal media (−control), treatment groups in basal media: 15 or 25%
DAMA extract, or complete growth media with 10% FBS (+ control).
The cells were then incubated for 24 h. After incubation, the bottom
side of the Transwell inserts were washed with PBS and fixed in 4%
paraformaldehyde for 20 min and stained with Hoechst 33342. Tile
scan images of 200 ×200 μm were acquired and migrated cells were
counted using ImageJ software (National Institutes of Health).
2.6.2 |Wound healing scratch assay
A wound healing scratch assay was performed to visualize cell migra-
tion. Passage 4 hDF were seeded at 7 ×10
4
cells per well into a
culture-insert 2 Well–24 well plate (Ibidi) and allowed to attach over-
night. 24 h later, the inserts were removed, cells were stained with
Hoescht 33342, and treatments were applied to the wells. Treatment
groups included basal media (−control), 15% DAMA extract in basal
media, 25% DAMA extract in basal media, and complete growth
media containing 10% FBS (+ control). Images were then taken at 0, 6,
20, and 27 h and analyzed with ImageJ software.
2.7 |Cellular proliferation assays
2.7.1 |BrDU cell proliferation assay
Passage 3 hDF were seeded at a density of 2.5 ×10
3
cells per cm
2
in
a 96-well plate. Treatment groups included: basal media with 1% FBS
(−control), 15 and 25% DAMA extract in basal media with 1% FBS, or
complete media with 10% FBS (+ control). After 24 h incubation with
treatment groups, BrdU reagent (BrdU Cell Proliferation ELISA, Roche)
was added to the wells and incubated for 24 h. Following incubation,
BrdU ELISA was performed according to the manufacturer's protocol
and plates were assayed in a plate reader (SpectraMax M3, Molecular
Device) at an absorbance of 450 and 690 nm.
2.7.2 |CyQuant cell proliferation assay
Passage 4 hDF (2 ×10
3
cells per cm
2
) were seeded in a 96-well plate
along with treatment groups: basal media with 1% FBS (−control), 15%
DAMA extract in basal media with 1% FBS or complete media with
10% FBS (+ control). Cells were cultured for up to 7 days with media
changes every 2 days. Timepoints were harvested at days 1, 3, and
7. CyQuant reagent (Thermo Fisher Scientific) was added to each well,
and the plates were read on a plate reader (SpectraMax M3) with fluo-
rescence setting at 480 nm excitation and 520 nm emission.
2.8 |Cell attachment and adhesion
Passage 4 hDF (5 ×10
4
cells per well) were seeded directly on DAMA
membranes (12 mm in diameter) and were cultured in basal media
3078 MOORE ET AL.
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with 1% FBS for 7 days. Cell seeded membranes were harvested at
days 1, 3, and 7 for image analysis. The membranes were washed with
PBS, fixed in 4% paraformaldehyde for 20 min and stained with
Hoechst 33342 and Alexa Fluor 488 Phalloidin for actin cytoskeleton
(Thermo Fisher Scientific).
2.9 |Inflammation inhibition assay
Human Peripheral blood mononuclear cells (PBMCS) were seeded at
1×10
5
cells per well in a 96-well plate and incubated for 1 h. Treat-
ment groups were incubated with PBMCS for 1 h and included 1 μM
Dexamethasone in RPMI media (−control), RPMI media with 10%
FBS (+ control), and 25% DAMA extract in RPMI media. PBMCs were
then stimulated with 100 pg/ml lipopolysaccharide (LPS from E. coli
bacteria) for 24 h. After 24 h, supernatants were collected for quanti-
fying TNF-α, IL-1β, and IL-6 levels using Luminex ELISA assay
(Millipore).
3|RESULTS
Growth factors, TIMPs, and cytokine concentrations in single layer
amniotic membranes were evaluated by use of a multiplex ELISA kit.
Some of the more notable factors present in the DAMA membranes
are bFGF, EGF, PDGF-AA, PDGF-BB, TGF-β, IL-6, IL-8, TIMP-1, and
TIMP-2 (Table 1).
Histological analysis was performed to visualize the membranes
as shown in Figure 1. An H&E stain reveals a single layer of amnion
with flattened epithelial cells. The ECM fibrous proteins appear dense
and compact since the membrane undergoes a dehydration step
during manufacturing. Immunostaining of DAMA membranes highlight
the presence of extracellular matrix proteins collagen type I, collagen
type III, fibronectin, and laminin.
Wound healing progression relies on the recruitment and prolifer-
ation of cells. Cellular recruitment and function are essential for the
deposition of new ECM proteins, signaling the recruitment of other
cells, angiogenesis, and regulation of inflammation. As shown in
Figure 2, fibroblast migration through a Transwell culture system
(Figure 2a,b) increases with the increasing concentration of DAMA
extracts (p< .05 for 25% DAMA vs (−) control). Similarly, the scratch
assay (Figure 2c,d), visually demonstrates increasing fibroblast migra-
tion in the wells containing DAMA extract compared to the (−) control
group (p< .05 for both 15 and 25% DAMA).
When cells migrate into a wound bed, it is important for them to
be able to proliferate to enable new tissue development. Figure 3
demonstrates that DAMA extracts influence fibroblast proliferation.
Furthermore, with increasing concentrations of extract, fibroblast pro-
liferation responds accordingly. Treatment with 15 or 25% DAMA sig-
nificantly increases fibroblast proliferation when compared to the (−)
control group (p< .0001) as detected by the BrDU cell proliferation
assay (Figure 3a). In addition, DAMA supports fibroblasts viability and
growth over 7 days in culture as shown in Figure 3b. Immunostained
images of cell-seeded DAMA membranes confirmed fibroblast attach-
ment onto the membranes and proliferation.
Cells and ECM interact in a synergistic manner with the matrix
providing a favorable environment for cell attachment, growth, prolif-
eration, and differentiation; in turn, the cells are able to secrete and
TABLE 1 Relative concentrations of growth factors present in
DAMA membranes as detected by ELISA
Concentration (pg/mg)
bFGF 1,113 ± 845
EGF 42.1 ± 7.1
G-CSF 1.99 ± 1.75
PDGF-AA 234.1 ± 15.2
PDGF-BB 38.3 ± 1.7
PLGF 16.9 ± 5.8
TGF-α1.9 ± 0.4
TGF-β1 41.5 ± 10.4
IL-4 0.7 ± 1.1
IL-6 1.71 ± 0.9
IL-8 10.7 ± 0.09
IL-10 0.33 ± 0.08
TIMP-1 4,746 ± 369
TIMP-2 841 ± 19
TIMP-4 16.1 ± 2.3 FIGURE 1 DAMA membrane was fixed and stained with H&E,
collagen type I, collagen type III, fibronectin, and laminin
MOORE ET AL.3079
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remodel the ECM. Figure 4 demonstrates that, in the presence of
DAMA extract, cells proliferate and secrete collagen type I. The cell
proliferation for groups with DAMA extract significantly increased as
compared to the (−) control group over 7 days in culture (p< .05). Col-
lagen type I levels increased by 7 days as confirmed by immuno-
staining (Figure 4a).
FIGURE 2 Evaluation of DAMA effect on fibroblast cell migration. (a, b) Transwell migration analysis of hDF stained with Hoechst after
migrating for 24 h (One-way ANOVA, *p< .05, ***p< .0002). (c) A scratch assay showing analysis of hDF migration into a void space at 0, 6,
20, and 27 h (Two-way ANOVA *p< .05)
FIGURE 3 Treatment of hDF
with 15 and 25% DAMA
increases cell proliferation as
detected by BrDU Cell
Proliferation Assay (One-way
ANOVA, ****p< .0001). (b) hDF
seeded on the surface of DAMA
membranes and cultured in basal
media for 1, 3, and 7 days and
stained for actin cytoskeleton
(green) and nuclei (blue)
FIGURE 4 Treatment of hDF with 15% DAMA increases ECM secretion and cell proliferation as detected by (a) Hoescht for nuclei (blue) and
collagen type I (green) stains at 7 days. (b) A DNA CyQUANT assay shows the cell number increases over time (Two-way ANOVA, **p< .01,
***p< .0002, ****p< .0001)
3080 MOORE ET AL.
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Chronic wounds persist with no wound resolution due to an ele-
vated inflammatory response within and surrounding the wound bed.
As demonstrated in Figure 5, a 25% DAMA extract significantly
decreased the level of pro-inflammatory cytokines secreted by LPS
stimulated PBMCs: TNF-α, IL-6 and IL-1β(p< .05 for cytokines). Spe-
cifically, IL-6 and TNF-αlevels were reduced by 83% and IL-1βlevels
were reduced by 70% when normalized to the (+) control group. It has
been shown that when these markers are down regulated with pla-
cental membranes, chronic wounds have a higher degree of wound
closure (Berven et al., 2010).
4|DISCUSSION
Chronic wounds, such as diabetic foot ulcers, that persist in the
inflammatory phase of healing can lead to morbidity and mortality if
not addressed (Ellis et al., 2018). Clinical application of DAMA was
previously shown to be beneficial for managing chronic diabetic foot
ulcers. Furthermore, one case series showed that wounds treated with
DAMA have a 60% decrease in wound size after the first application
(Boyar & Galiczewski, 2018). As previously presented by Snyder
et al. (2016), DAMA accelerates diabetic foot ulcer wound closure
with 45% of patients achieved complete wound closure as compared
to 0% of the wounds treated with the standard of care having closure
by 6 weeks. We hypothesize that DAMA aides in the wound healing
process by releasing growth factors, which then regulates cell behav-
ior in the wound microenvironment. The objective of this study was
to characterize growth factor content in DAMA and their ability to
influence in vitro cell functions specific to wound healing. The results
demonstrate that DAMA consisted of multiple growth factors and
cytokines, which are known to participate in the wound healing pro-
cess. In addition, DAMA is a bioactive membrane that positively
impacts hDF activity in vitro.
In normal wound healing, there is an interplay between several
cell types including fibroblasts, keratinocytes, and inflammatory cells
along with the surrounding ECM, also known as dynamic reciprocity.
These cells interact by secreting growth factors, cytokines, and ECM
that guide tissue remodeling through several different phases. DAMA
is a rich source of cytokines and growth factors as demonstrated by
ELISA results. Some of the factors present in these membranes
providing clinical benefits include EGF, bFGF, PDGFs, TGF-α, and
TIMPs 1 and 2. EGF has a role in the stimulation, proliferation, and
migration of many different cell types including hDF, keratinocytes,
and endothelial cells and has been shown to accelerate healing
(Brown et al., 2010). bFGF has multiple biochemical and biological
roles including angiogenesis, proliferation and migration of many cell
types, stimulation of collagen synthesis, and epithelialization (Grazul-
Bilska et al., 2003). PDGF is involved in all stages of wound healing
and has a role in the chemotaxis of hDF and smooth muscle cells,
stimulation of collagen and glycosaminoglycans production from fibro-
blasts, and signals to other cells including the keratinocytes in the epi-
thelial layer (Koob et al., 2014b; Lynch et al., 1987). TIMP 1 and
2 both have a role in regulating several matrix metalloproteinases
(MMPs). MMPs are involved in many phases of wound healing such
as epithelialization and inflammatory responses and can act in both a
positive and negative manner during wound healing (Gill &
Parks, 2008).
When wounds remain in the inflammatory phase, high levels of
upregulated cytokines persist in the wound bed such as: IL-6, IL-8,
IL-1 β, and TNF-α. IL-6 promotes angiogenesis, however at high levels,
IL-6 has been known to delay re-epithelialization (Lin et al., 2003). In a
chronic wound TNF-αand IL-1βhave a synergistic effect in promoting
the release of MMPs and suppressing the synthesis of TIMPs and
ECM forming proteins (Barrientos et al., 2008; Xu et al., 2013). IL-8
expression aides in the stimulation of neutrophil migration which
induces an inflammatory response at the wound site. However, over
simulation of IL-8 can lead to destruction of healthy ECM by neutro-
phils, preventing wounds from entering the proliferative stage (Ellis
et al., 2018).
A reduction in pro-inflammatory cytokines has been linked to the
progression of chronic wounds through the healing process and ulti-
mately wound closure. High levels of pro-inflammatory cytokines
inhibit healing capabilities; thus, the management of these cytokines
is essential to the treatment of stalled wounds.
DAMA is shown to reduce pro-inflammatory cytokines IL-1β,
IL-6, and TNF-αand when reduced will allow the wound to progress
into the proliferation stage. In the proliferative stage, hDF will migrate
and proliferate, becoming the predominant cell type in the wound
(Stadelmann et al., 1998). Once in the wound bed, hDF secrete differ-
ent types of ECM such as collagens and GAGs to fill the wound bed
FIGURE 5 Reduction in pro-inflammatory cytokines secreted from PBMCs in the presence of 25% DAMA as compared to an LPS-treated
positive control (One-way ANOVA, **p< .002, ***p< .001, ****p< .0001)
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while supporting epithelialization and angiogenesis (Dobaczewski
et al., 2010). Furthermore, as demonstrated in vitro, DAMA promotes
migration and cell proliferation of hDF which further supports the
wound transition from the inflammatory to proliferative phase. This
cascade of wound resolution occurs once the appropriate environ-
ment for wound healing is created and DAMA membranes have the
ability to restore favorable cellular responses for proper wound
healing.
Placental tissue membranes have gained traction in the chronic
wound space because of their ability to progress and resolve difficult
to heal wounds. As many studies have shown, using amniotic mem-
branes decreases scar tissue, reduces anti-inflammatory markers,
increases expression levels of angiogenic proteins, and can assist with
re-epithelialization (Adzick & Longaker, 1992; Hao et al., 2000; Mer-
met et al., 2007). These are all factors that have an influence in creat-
ing the proper balance of factors for proper wound healing (Tseng
et al., 1999). Unlike traditional collagen matrices, DAMA membranes
have a role in regulating the inflammatory response and assisting in
the progression of chronic wounds. While many factors that contrib-
ute to the wound healing benefits of amniotic membranes are dis-
cussed, future studies will investigate other possible factors of pro-
inflammatory cytokine reduction as well as differences with traditional
ECM scaffolds in a wound healing model.
CONFLICT OF INTEREST
Funding for this project was provided by Integra LifeSciences.
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How to cite this article: Moore MC, Bonvallet PP,
Damaraju SM, Modi HN, Gandhi A, PS McFetridge. Biological
characterization of dehydrated amniotic membrane allograft:
Mechanisms of action and implications for wound care.
J Biomed Mater Res. 2020;108B:3076–3083. https://doi.org/
10.1002/jbm.b.34635
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