Comparison of cryopreserved amniotic membrane and umbilical cord tissue with dehydrated amniotic membrane/chorion tissue

Abstract

Objective:
To evaluate how the different processing methods cryopreservation and dehydration affect the structural integrity and biological composition of key signalling molecules within amniotic membrane and umbilical cord tissues.

Method:
We directly compared cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues with dehydrated amniotic membrane/chorion (dHACM) tissue using biochemical and functional assays including histological and histochemical staining, BCA, agarose gel electrophoresis, western blot, ELISA, and proliferation and cell death assays.

Results:
Cryopreservation retains the native architecture of the AM/UC extracellular matrix and maintains the quantity and activity of key biological signals present in fresh AM/UC, including high molecular weight hyaluronic acid, heavy chain-HA complex, and pentraxin 3. In contrast, dehydrated tissues were structurally compromised and almost completely lacked these crucial components.

Conclusion:
The results presented here indicate that cryopreservation better preserves the structural and biological signaling molecules of foetal tissues.

Full text

THIS ARTICLE IS REPRINTED FROM THE JOURNAL OF WOUND CARE VOL 23, NO 10, OCTOBER 2014
M. Cooke,1 BS, Associate Product Manager; E.K. Tan,2, 3 MS Director of Product Development;
C. Mandrycky,4 BSE, Laboratory Technician; H. He, 2, 3 PhD, is a Senior Scientist; J. O’Connell,1 PhD, Senior Director of
Research and Development; S.C.G. Tseng,1,2,3 MD, PhD, Chief Scientic Ofcer;
1 Amniox Medical, Atlanta, GA, 30339, USA; 2 TissueTech, Inc., Miami, FL, 33173, USA;
3 Ocular Surface Center, Miami, FL, 33173, USA; 4 Wallace H. Coulter Department of Biomedical Engineering
at Georgia Institute of Technology, Atlanta, GA, 30332, USA.
Email: stseng@ocularsurface.com
journal of wound care
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volume 23. number 10. october 2014
Comparison of cryopreserved
amniotic membrane and umbilical
cord tissue with dehydrated
amniotic membrane/chorion tissue

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© 2014 MA HeAltHcAre ltd
Comparison of cryopreserved amniotic
membrane and umbilical cord tissue
with dehydrated amniotic membrane/
chorion tissue
l Objective: To evaluate how the different processing methods cryopreservation and dehydration affect
the structural integrity and biological composition of key signalling molecules within amniotic membrane
and umbilical cord tissues.
l Method: We directly compared cryopreserved amniotic membrane (AM) and umbilical cord (UC)
tissues with dehydrated amniotic membrane/chorion (dHACM) tissue using biochemical and functional
assays including histological and histochemical staining, BCA, agarose gel electrophoresis, western blot,
ELISA, and proliferation and cell death assays.
lResults: Cryopreservation retains the native architecture of the AM/UC extracellular matrix and
maintains the quantity and activity of key biological signals present in fresh AM/UC, including high
molecular weight hyaluronic acid, heavy chain-HA complex, and pentraxin 3. In contrast, dehydrated
tissues were structurally compromised and almost completely lacked these crucial components.
lConclusion: The results presented here indicate that cryopreservation better preserves the
structural and biological signaling molecules of foetal tissues.
lDeclaration of interest: S.C.G. Tseng and his family are more than 5% shareholders of TissueTech,
Inc. TissueTech, Inc. and its Subsidiaries (Bio-Tissue and Amniox Medical) own US Patents Nos. 6,152,142;
6,326,019; and PCT/US2010/046675 on the method of preparation and clinical uses of human amniotic
membrane with the CryoTek method distributed by Bio-Tissue, Inc. and Amniox Medical. S.C.G. Tseng,
E.K. Tan, and H. He are employees of TissueTech, Inc. J. O’Connell and M. Cooke are employees and
shareholders of Amniox Medical. C. Mandrycky has no nancial conict. This research was supported by
a Venture Lab Grant #398 from the Georgia Research Alliance, Atlanta, GA, and a research grant from
TissueTech, Inc., Miami, FL.
amniotic membrane, umbilical cord, cryopreserved, dehydrated, anti-inflammatory, anti-scarring
A
mniotic membrane (AM) consists of a
monolayer of simple epithelium
attached to a thick basement mem-
brane and an underlying avascular stro-
mal region that can be further subdi-
vided into compact, broblast and spongy layers.
The stroma of the umbilical cord is primarily com-
posed of a viscous connective material called Whar-
ton’s jelly surrounded by an outer layer of AM. While
the umbilical cord supplies the foetus with oxygen-
ated, nutrient-rich blood, the AM protects the foetus
from maternal insults during development.1,2
Clinically, AM has been used as an allograft across
multiple disciplines. Particularly in ophthalmology,
transplantation of AM has become a standard surgi-
cal procedure for ocular surface reconstruction to
promote epithelialisation and reduce inammation
and scarring.3 Elsewhere, cryopreserved amniotic
tissue has been used to treat wounds in different
types of tissue including tendon4,5 and nerve
repair.6,7 AM was used to cover open wounds as early
as 1910,8 and has also been used on wounds caused
by many other aetiologies such as venous leg
ulcers,9–12 pressure ulcers9,13–15, diabetes mellitus,15
trauma15,16 and burns.17–22
While the clinical use of AM is well documented,
the use of umbilical cord (UC) tissue in this setting
is relatively new. Comprised of AM with additional
components unique to the foetal environment, it is
likely that UC would have comparable, if not
increased, effectiveness to AM in both biological
and functional assays and is therefore included in
the analyses presented here.
The successful commercialisation of any tissue
product relies on the effective preservation of key
biological components essential to maintaining the
intended therapeutic action of the tissue. Differ-
ences in processing methods can dramatically alter
both the structural and biochemical composition of
the tissue, and impair the activity of vital signalling
M. Cooke,1 BS, Associate
Product Manager;
E.K. Tan,2, 3 MS Director
of Product Development;
C. Mandrycky,4 BSE,
Laboratory Technician; H.
He, 2, 3 PhD, is a Senior
Scientist; J. O’Connell,1
PhD, Senior Director of
Research and
Development; S.C.G.
Tseng,1,2,3 MD, PhD,
Chief Scientic Ofcer;
1 Amniox Medical,
Atlanta, GA, 30339, USA;
2 TissueTech, Inc., Miami,
FL, 33173, USA; 3 Ocular
Surface Center, Miami, FL,
33173, USA; 4 Wallace H.
Coulter Department of
Biomedical Engineering at
Georgia Institute of
Technology, Atlanta, GA,
30332, USA.
Email: stseng@
ocularsurface.com

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molecules such as cytokines, proteoglycans and
growth factors, which are critical to the intended
use of the product.23 From AM, we have successfully
puried and characterised ‘HC-HA/PTX3’ as a
unique matrix component responsible for its thera-
peutic actions.24–28,30 HC-HA/PTX3 is formed by
tight association between pentraxin 3 (PTX3), a
member of the pentraxin family of proteins, known
to be involved in innate immunity, and HC-HA, a
high molecular weight (HMW) hyaluronic acid (HA)
covalently linked to heavy chain 1 (HC1) of inter-
α-trypsin inhibitor (IαI).24–28,30 In vitro, soluble HC-
HA/PTX3 suppresses the proliferation and promotes
apoptosis of lipopolysaccharide (LPS)-induced mac-
rophages.27 Additionally, immobilised HC-HA/PTX3
upregulates IL-10 and downregulates IL-12 upon
stimulation of IFN-α and LPS to polarise macro-
phages toward the M2 (activated macrophage) phe-
notype which plays an integral role in promoting
the healing response.27-29 Therefore, measurement
of the presence and function of HC-HA/PTX3 can
be used to judge how well different processing
methods may preserve the therapeutic actions of
AM and UC.
While several processing methods exist, the most
widely employed are cryopreservation and dehydra-
tion via heat drying. In order to judge the effects of
cryopreservation on AM and UC tissues, we recently
reported that cryopreserved AM/UC is comparable to
fresh AM/UC based on the retention of key architec-
tural and biochemical components essential for the
therapeutic actions of the tissues.31 Here, we extend
our study to compare cryopreserved AM/UC tissues
with dehydrated amnion/chorion tissues in order to
verify whether different processing methods might
affect tissue integrity and therapeutic potential.
Materials and methods
Tissue preparation
Cryopreserved human AM (CT-AM) and UC (CT-
UC) tissue processed by the CryoTek (CT) method
(US 6,326,019, US 6,152,142 and PCT/
US2010/046675) was provided by TissueTech, Inc.
(Miami, FL) and compared with dehydrated tissue
(dHACM), processed by the PURION method (US
8,323,701).To prepare cryopreserved AM and UC tis-
sues, donated full-term human placentas with the
umbilical cord were recovered after cesarean-section
delivery in compliance with American Association
of Tissue Banks (AATB) standards and immediately
stored at -80°C for up to one year. Prior to process-
ing, the frozen placenta and UC were thawed at
room temperature for 8 hours in a Good Manufac-
turing Practice (GMP) facility before being placed at
8°C for an additional 16 hours. Under aseptic condi-
tions, the placenta and UC were rst cleaned of
blood clots with phaosphate-buffered saline (PBS)
prior to separation of AM and UC by blunt dissec-
tion. The chorion was separated from AM and blood
vessels were stripped from UC to generate a at graft
before gentle rinsing in PBS until all blood colora-
tion was removed. AM was afxed on a lter mem-
brane and cut to 6 x 6cm while UC was cut to 6 x
3cm. The AM or UC tissue was nally packaged in a
pouch containing 1:1 v/v Dulbecco Modied Eagle
Medium (DMEM) and glycerol before storage at
-80°C for up to two years.
Histology and histochemistry
Cryopreserved tissues were allowed to thaw for 10
minutes at room temperature and dehydrated sam-
ples were rehydrated according to package instruc-
tions. All tissues were xed with 10% formalin for
one hour, washed three times with PBS for ve min-
utes each, and cut with a 15mm biopsy punch to
obtain equal-sized tissue samples. Tissues were sub-
sequently embedded in histogel, processed, embed-
ded into parafn blocks, and cut into 5μm sections.
Histological sections were then stained with either
hematoxylin & eosin (H&E), Masson’s trichrome
(MAS), or Safranin O (SafO) with Fast Green FCF
counterstain. For HA histochemistry, de-parafn-
ised sections were incubated with biotinylated
Hyaluronic Acid Binding Protein (HABP) followed
by Alexa Fluor 488 streptavidin. Nuclei were coun-
terstained with Hoechst-33342 and images were
photographed by laser confocal microscopy
(LSM700, Axio Observer.Z1, Zeiss, Germany).
Tissue-extract preparation
The preparation of tissue extracts for CT-AM, CT-UC
and dHACM was carried out aseptically as previ-
ously reported.32 To assess the retention of key bio-
chemical molecules, homogenised tissues were
extracted by 4M guanidine hydrochloride and the
supernatant dialysed against PBS for 30 hours to
obtain water-soluble extracts from each tissue. Pro-
tein content was quantied using a standard BCA
assay (Pierce) while quantication of HA was per-
formed by HABP-coated microwell kit (Corgenix).
Determination of HA sizes by agarose-gel
electrophoresis
The molecular weight of HA in tissue extracts was
analysed by agarose gel electrophoresis as previous-
ly reported.33 Tissue extracts were loaded at the same
equivalent (15µg) of HA per lane with or without
pretreatment (one hour, 37°C) with 9 units HAase
per μg. The samples were separated on a 0.5% agar-
ose gel at 20V for the rst 30 minutes and then 40V
for 4 hours. The gel was subsequently stained with
0.005% Stains-all dye in 50% ethanol overnight at
25°C in the dark before destaining in water and
exposing to ambient light for 6 hours. The molecu-
lar weight range of HA samples, which appear as a

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bluish smear on the agarose gel, was estimated by
comparison to the Select-HA HiLadder and HMW
HA (Healon).
Western blot
Tissue-extract samples were loaded with equivalent
(20µg) protein per lane with or without pretreat-
ment (one hour, 37°C) with two units of HAase per
μg HA before denaturation in Laemmli Buffer (1:1
dilution with sample) at 95°C for ve minutes. Sam-
ples were then electrophoresed and electrophoreti-
cally transferred on to a 0.45µm nitrocellulose
membrane. The membrane was then blocked with
5% fat-free milk in tris-buffered saline and tween 20
(TBST) and sequentially incubated with mouse anti-
human primary antibodies against HC1 (1:1000) in
5% fat-free milk in TBST (16 hours, 4°C) followed by
rabbit anti-mouse HRP-conjugated secondary anti-
body (1:1000) in 5% fat-free milk in TBST (two
hours, room temperature). Immunoreactive protein
bands were detected with Western Lightning
Chemiluminesence Reagent and imaged by a Lumi-
nescent Image Analyzer (ImageQuant LAS 4000,
GE). The same membrane was rinsed and stripped
with Restore Plus Western Blot Stripping Buffer and
then re-blocked with 5% fat-free milk in TBST and
sequentially incubated with rat anti-human prima-
ry antibodies against PTX3 (1:1000) in 5% fat-free
milk in TBST (16 hours, 4°C) followed by goat anti-
rat HRP-conjugated secondary antibody (1:1000) in
5% fat-free milk in TBST (2 hours, room tempera-
ture). Immunoreactive protein bands were detected
with Western Lighting Chemiluminesence Reagent
and imaged by a Luminescent Image Analyzer.
Macrophage proliferation assay
RAW264.7 cells were seeded at a density of 156 cells/
mm2 on a 96-well plate in DMEM/10% foetal bovine
serum (FBS) (t=0) and treated at t=2 hours with
either CT-AM, CT-UC, or dHACM tissue extracts
containing 100μg protein/ml, or PBS vehicle con-
trol before activation with IFN-α (200units/ml) and
LPS (1μg/ml) at t=3 hours. At t=25 hours, cells were
labelled with 10µM BrdU for 2 hours and were sub-
sequently xed with FixDenat (provided in BrdU
ELISA kit, Roche) at 25°C for 30 minutes, followed
by incubation with anti-BrdU-peroxidase conjugate
at 25°C for 2 hours. The colour was developed for 30
minutes by adding the substrate tetramethylbenzi-
dine (TMB) and stopped by adding 1M H2SO4.
Colorimetric measurements were performed at
450nm with a reference wavelength at 690nm.
Macrophage cell death assay
RAW264.7 cells were seeded and treated as above for
the rst three hours. At t=27 hours, cell lysates were
collected using lysis buffer (provided in Cell Death
ELISA kit, Roche). The cell lysate (20μl) was trans-
ferred to a microplate and 80μl of Immunoreagent
was added before incubation at 300rpm (2 hours,
25°C). After incubation, wells were rinsed three
times with 300μl incubation buffer. The colour was
developed for 20 min by adding 100μl of ABTS solu-
tion and stopped by adding 100μl ABTS Stop Solu-
tion. Colorimetric measurements were performed at
405nm with a reference.
ELISA analysis
RAW264.7 cells were seeded at a density of 250 cells/
mm2 on a 24-well plate in DMEM/10% FBS (t=0)
and treated at t=2 hours with either CT-AM, CT-UC,
or dHACM tissue extracts containing 100μg pro-
tein/ml, or PBS vehicle control before activation
with IFN-α (200 units/ml) and LPS (1μg/ml) at t=3
hours. Culture media from each well was collected
at t=27 hours for IL-10 and IL-12 ELISA analysis.
Briey, a capture antibody was adsorbed onto a
96-well plate, followed by a blocking step, incuba-
tion with 100µL sample, and binding of analyte to a
biotinylated detection antibody. Concentrations of
capture and detection antibodies were used accord-
ing to the manufacturer’s protocol (BioLegend).
IL-10 or IL-12 in the cell supernatants was assessed
using the colorimetric reaction of peroxidase TMB
at an absorbance reading of 450nm.
Statistical analysis
Unless otherwise indicated, data are represented as
mean±standard error with a sample size of three or
more for each condition. A Student’s t-test was per-
formed to test for statistical signicance in protein
levels with Microsoft Ofce Excel 2007. An analysis
of variance (ANOVA) coupled with Tukey’s post-hoc
analysis was performed to test statistical signicance
for HA quantication, macrophage cell prolifera-
tion, macrophage cell death and ELISA analysis with
SPSS Statistics 20 (IBM). Where p<0.05 the results
were considered statistically signicant.
Results
Histological and immunohistochemical staining
To compare the effects of cryopreservation and
dehydration on tissue morphology and extracellular
matrix (ECM) components, sections were examined
by histochemical staining We have previously
shown that cryopreserved AM/UC tissues contain
no signicant structural differences compared with
fresh tissues.31 As seen previously, H&E staining
revealed that the structural morphology of CT-AM
(Fig 1a) closely resembled that of fresh AM tissue,
which contains a thin basement membrane sand-
wiched between a simple epithelium and an avascu-
lar stroma. As expected, CT-UC mirrored this mor-
phology, with an expanded stromal layer (Fig 1b). In
contrast, dHACM contained a layer of chorion in

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addition to the AM layer, and was dramatically
compacted in comparison to the cryopreserved tis-
sue (Fig 1c). Both Masson’s Trichrome (Fig 1 d–f)
and Safranin O staining (Fig 1 g–i), used to indicate
ECM collagen content and sulfated proteoglycans,
respectively, corroborated these results. Collectively,
these ndings support the notion that cryopreserva-
tion did not cause notable changes in the histologi-
cal properties of thin or thick AM tissue, while
dehydration drastically altered the structural integ-
rity of the tissue.
Biochemical analyses of HA and HC-HA
Recent studies have indicated HC-HA/PTX3 is a key
signalling proteoglycan present in AM/UC.25–28,30,31
Histochemistry with HABP was performed to visual-
ise the density and distribution of HA within the
ECM of cryopreserved AM/UC and dHACM. HA
staining was positive in the AM stroma but absent
in the amniotic epithelium for all tissues. Of note,
HA was only weakly present in the subjacent chori-
on of dHACM. Cryopreserved tissues presented
robust uniform staining across the stromal layer,
especially apparent in the thick stromal layer of the
CT-UC (Figs 2 a and b,). dHACM exhibited only
modest HA staining that was sporadically distribut-
ed throughout the ECM, indicating the disruption
and removal of HA originally present in the mem-
brane by this processing method (Fig 2c). The
absence of staining following pretreatment of histo-
logic sections with HAase conrmed the specicity
of HA staining (Fig. 2 d–f).
To directly quantify the differences in ECM HA
content, tissue extracts were analysed using an
HABP assay. While both cryopreserved samples
exhibited an increased HA/total protein ratio com-
pared to dHACM, the results only reached signi-
cance in the CT-UC sample (Fig 3). Consistent with
HA staining, the CT-UC sample contained signi-
cantly more (~50X) HA than dHACM. Of note,
while not signicantly different, CT-AM contained
three times more HA than the dehydrated tissue,
likely due to the chorion layer contributing to an
increase in total protein while appearing to have
comparatively little HA.
The molecular weight of HA has been tied to dif-
ferent biological outcomes in vivo with critical dis-
tinction between low molecular weight HA (LMW
HA) and high molecular weight (HMW) HA.33 To
determine the size distribution of HA in the tissue
extracts, we used agarose gel electrophoresis fol-
lowed by Stains-all dye as previously described.25
Staining was shown to be specic for HA by the dis-
appearance of the HMW HA fraction with HAase
digestion (Fig 4, lanes 6–8). HA from CT-UC (Fig 4,
lane 4) exhibited a similar HMW distribution as the
Fig 2. HA histochemistry of cryopreserved and dehydrated tissues. Tissues
were incubated with HA binding protein with (d,e,f) or without (a,b,c)
HAase digestion. All images are 10x magnication, scale bar=200µM.
a
d
b
e
c
f
CT-AM CT-UC dHACM
HAase +
HAase -
HA=hyaluronic acid, CT-AM=cryopreserved amniotic membrane, CT-UC=cryopreserved umbilical cord,
dHACM=dehydrated human amnion/chorion membrane
Fig 3. HA content quantied using a HABP
microwell kit and normalised to total protein. The
amount of HA retained in CT-UC was signicantly
higher than all other tissues.
HA (g)/Protein (g)
CT-AM CT-UC dHACM
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
*
* Indicates p<0.01. CT-AM=cr yopreserved amniotic membrane,
CT-UC=cryopreserved umbilical cord, dHACM=dehydrated human amnion/
chorion membrane
Fig 1. Histological staining of cryopreserved and dehydrated amniotic
tissues. CT-AM (a, d ,g), CT-UC (b, e, h) and dHACM (c, f, i). All images are
20x magnication.
a
d
g
b
e
h
c
f
i
CT-AM CT-UC dHACM
CT-AM=cryopreserved amniotic membrane, CT-UC=cyropreserved umbilical cord, dHACM=dehydrated human
amnion/chorion membrane
Hematoxylin and
eosin
Masson’s Trichrome
Safranin O

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Healon control (Fig 4, lane 2) in the range of 1000 to
6000 kDa, indicating that UC preferentially con-
tained HMW HA. In contrast, dHACM contained no
HMW HA, instead displaying a LMW HA smear at
the bottom of the gel (Fig 4, lane 5) with a molecular
weight below 500 kDa. Interestingly, a HMW band
was detected in the loading wells of both CT-AM and
CT-UC samples (Fig 4, lanes 3 and 4) which was abol-
ished after HAase digestion (Fig 4, lanes 6 and 7), sug-
gesting the presence of a HMW HA species in cryop-
reserved AM/UC that was larger than that of Healon.
In contrast, no band from the dehydrated sample
existed in the loading well (Fig 4, lane 5), corroborat-
ing the LMW smear within the lane.
Previous research has shown that the HA present
in AM/UC exists as a HC-HA/PTX3 complex.25–28,30,31
To determine if this complex was altered or
destroyed by the dehydration processing method,
we subjected tissue extracts from cryopreserved and
dehydrated tissues to Western blot using an HC1-
specic antibody. Additionally, to ensure that HC1
was indeed associated with HA, tissue-extract sam-
ples were examined in parallel by rst treating one
replicate with NaOH, which hydrolyses the ester
bond between HA and HC1. As expected, IαI puri-
ed from human plasma yielded a major band at
~250kDa (Fig 5, lane 2) and treatment of IαI with
50mM NaOH cleaved the ester bonds linking the
HCs to IαI to yield a 75kDa HC1 fragment (Fig 5,
lane 3). As seen in the agarose gel, both cryopre-
served tissues contained a band that remained in
the well (Fig 5, lanes 5 and 7) most likely HC-HA,
because digestion with NaOH partially cleaved the
complex to yield an increase in the intensity of the
75kDa HC1 fragment (Fig. 5, lanes 6 and 8) indicat-
ing that the HC-HA complex is present in both CT-
AM and CT-UC tissues. Interestingly, a band
remained in the CT-UC well even after digestion
with NaOH, possibly due to the large amount of HA
within the CT-UC extract. The dehydrated sample
did not contain any HMW bands and exhibited the
75kDa HC1 band before and after digestion, sug-
gesting the lack of the HC-HA complex due to the
degradation and removal of HMW HA, leaving only
unbound LMW HA.
PTX3 content
PTX3 is an oligomeric protein shown to be consti-
tutively expressed in the AM that serves to stabilise
the HC-HA complex. 30,34 In order to determine the
effects of processing methods on PTX3 protein lev-
els, tissue extracts were analysed via Western blot.
PTX3 exists as two forms: a ~45kDa monomer and
~90kDa dimer (Fig 6, lane 4). Cryopreserved tissue
extracts had strong PTX3 bands at 45 and 90kDa
(Fig 6, lanes 5–8). In addition, the CT-UC sample
contained another band that remained in the well
due to its large size (Fig 6, lanes 7 and 8), similar to
Fig 4. Agarose gel electrophoresis of CT-AM, CT-UC and dHACM tissue
extracts was performed to determine the size of HA present. Select-HA
HiLadder (M, Lane 1) and HMW HA control (Lane 2). The specicity of HA
was conrmed by HAase digestion (Lanes 6–8)
M
1
HA
2
CT-AM
3
CT-UC
4
dHACM
5
HAase (–) HAase (+)
CT-AM
6
CT-UC
7
dHACM
8
Lane
kDa
6100
4570
3050
1520
1090
966
572
495
HA=hyaluronic acid, HMW=high molecular weight, CT-AM=cryopreserved amniotic membrane
CT-UC=cryopreserved umbilical cord, dHACM=dehydrated human amnion/chorion membrane
Fig 5. Heavy chain hyaluronic acid complex detected by Western Blot.
HC-HA
IαI
HC1
kDa
200
140
100
80
60
50
40
30
20
10
HC1 NaOH
IαI
– +
2 3
CT-AM
– +
5 6
CT-UC
– +
7 8
dHACM
– +
9 10
PTX3
–
4
M
1
CT-AM=cryopreserved amniotic membrane CT-UC=cryopreserved umbilical cord, dHACM=dehydrated human
amnion/chorion membrane, PTX3= pentraxin 3, HC-HA= Heavy chain hyaluronic acid, HC1=heavy chain 1

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the agarose gel, indicating the presence of PTX3 as
part of the HC-HA complex. In comparison, the
dehydrated sample was devoid of PTX3 (Fig 6,
lanes 9 and 10), supporting the idea that the dehy-
dration process degrades and removes PTX3 natu-
rally present in the ECM of AM while cryopreserva-
tion preserves the integrity of this critical HC-HA
stabilising protein.
Functional analyses
AM’s anti-inammatory activity has been demon-
strated by the suppression of viability and prolifera-
tion of macrophages by both AM tissue35,36 and AM
extracts.3,32 To explore the effect of processing on
innate activity, tissue extracts containing 100µg
protein/ml from either cryopreserved or dehydrated
samples were applied to activated RAW264.7 cells,
(a macrophage cell line) and both macrophage pro-
liferation (Fig 7a) and cell death (Fig 7b) were meas-
ured in separate assays. Compared with cells in the
PBS vehicle control, RAW264.7 macrophages treat-
ed with cryopreserved tissue extracts showed signi-
cantly inhibited proliferation (p<0.01; ANOVA fol-
lowed by Turkey’s post-hoc test). However, tissue
extract from dehydrated samples was comparable to
the control and displayed no signicant effect. Fur-
thermore, only CT-UC was signicantly (p<0.01;
ANOVA followed by Turkey’s post-hoc test) more
effective than the control in promoting macrophage
apoptosis. In fact, CT-UC-stimulated apoptosis was
approximately 14 times more greater than that seen
with all other tissue extracts.
ELISA quantication of cytokines
We have previously reported that the HC-HA/PTX3
complex downregulates pro-inammatory cytokines
while upregulating anti-inammatory
cytokines.27,28,30 ELISA analysis of two critical
cytokines IL-10 (anti-inammatory) and IL-12 (pro-
inammatory) was performed to elucidate what
effects, if any, processing techniques had on HC-
HA/PTX3 ability to regulate cytokine expression.
CT-UC extract dramatically increased IL-10 expres-
sion compared to all other samples, with IL-10 lev-
els approximately 8-fold higher than dehydrated
samples (Fig 8a). When assessing IL-12 expression,
all samples were signicantly different to control,
with CT-AM and dHACM exhibiting similar expres-
sion levels and CT-UC reducing IL-12 expression 7–
fold more than dHACM (Fig 8b).
Discussion
Fresh AM has been proven efcacious in clinical
applications.11,37,38 However, its use presents a seri-
ous risk of disease transmission.39,40 Therefore,
processing methods that ensure the safety of the tis-
sue while preserving its innate biological effective-
ness become critical. With several processing proto-
cols commonly used, techniques differ dramatically
and can have varying impacts on both the structur-
al and biochemical composition of the tissue, as
well as the activity of critical signalling molecules
(cytokines, proteoglycans, etc), essential to the ther-
apeutic actions of the tissue.23 Specic to the meth-
ods compared here, the cryopreservation process
involves quickly freezing the tissue and was speci-
cally developed to maintain the structural integrity
of the extracellular matrix and the endogenous bio-
chemical functions of the native AM and UC tis-
sues. In contrast, dehydration is much harsher and
has been shown to cause protein denaturation, loss
of function, and irreparable damage to the
ultrastructure and material properties of the tissue,
resulting in loss of cell attachment and decreased
cell inltration.23,41–43 This process creates the poten-
tial for compromising the survival and permanence
of key anti-inammatory and anti-scarring factors,
suggesting the clinical efcacy of tissue processed by
this method may be lessened.
Histological analysis revealed that the ECM archi-
tectural structure within CT-AM and CT-UC was not
altered by cryopreservation but was compromised
by dehydration. This is a critical nding, as the pro-
teins within the ECM regulate the functions of cells
and small molecules, and act as a reservoir and
modulator of cytokines and growth factors.44,45
Moreover, the healing potential of AM is mediated
by the complex assembly of these components,46–48
further indicating the importance of maintaining
the integrity of the ECM after preservation. Of note,
cryopreserved tissues were comparable in structure
Fig 6. Pentraxin 3 identied by Western Blot.
HC-HA
PTX3
(dimer)
PTX3
(monomer)
kDa
200
140
100
80
60
50
40
30
20
10
PTX3 NaOH
IαI
– +
2 3
CT-AM
– +
5 6
CT-UC
– +
7 8
dHACM
– +
9 10
PTX3
–
4
M
1
CT-AM=cryopreserved amniotic membrane, CT-UC=cryopreserved umbilical cord, dHACM=dehydrated human
amnion/chorion membrane, PTX3= pentraxin 3, HC-HA= heavy chain hyaluronic acid

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to fresh AM31 while dehydrated tissues were notably
impaired, indicated by the compact appearance of
the stromal and chorion layers.
The clinical success of AM/UC as a potent anti-
inammatory and anti-scarring agent has prompted
examination into the physiological mechanism of
its therapeutic actions. Previous studies revealed
AM’s regenerative potential may be mediated by
complex arrays of cytokines, chemokines, and
growth factors which have been linked to the pro-
motion of wound healing via suppression of host
immune cells.49,50 Recent studies have implicated HA
complex as a critical biological component that con-
tributes to the anti-inammatory and anti-scarring
properties of AM.25,28,30,32 Cryopreservation retained
high levels of HA within both CT-AM and CT-UC
samples and also preserved the distribution of HA
across the stromal layer of the tissues. In contrast,
while HA was still present in the dHACM sample,
the distribution was radically altered, with sporadic
pockets of HA randomly dispersed throughout the
tissue and total levels of HA much lower, compara-
tively. Moreover, the HA present within cryopre-
served tissues, particularly CT-UC, was HMW HA,
while the HA contained in dHACM was LMW HA.
This is of particular interest because certain studies
have suggested HMW HA is the key isoform of HA
responsible for the therapeutic properties mentioned
above while LMW HA contributes to the inamma-
tory response and is immune-stimulatory.33,51
Fig 7. Effects of tissue extracts on macrophage proliferation (a) and cell death (b).
Absorbance/Protein
CTL CT-AM CT-UC dHACM
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
*
*
Absorbance/Protein
CTL CT-AM CT-UC dHACM
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
*
a b
* Indicates p<0.01 compared to all other groups. CT-AM=cryopreserved amniotic membrane, CT-UC=cr yopreserved umbilical cord, dHACM=dehydrated
human amnion/chorion membrane, CTL=control.
Fig 8. Macrophage cytokine expression in response to tissue extracts. IL-10, an anti-inammatory cytokine (a)
and IL-12, a pro-inammatory cytokine (b).
Absorbance/Protein
CTL CT-AM CT-UC dHACM
6.0
5.0
4.0
3.0
2.0
1.0
0.00
*
Absorbance/Protein
CTL CT-AM CT-UC dHACM
0.10
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
*
p=0.010
ab
* Indicates p<0.01 compared to all other groups. CT-AM=cryopreserved amniotic membrane, CT-UC=cr yopreserved umbilical cord, dHACM=dehydrated human
amnion/chorion membrane, CTL=control.

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We have previously shown that HMW HA in AM
exists within the HC-HA complex that is stabilised
by the multimeric protein PTX325,26,28,30 and that this
larger complex exerts a more powerful therapeutic
effect than HMW HA alone.27,28 Western blot analy-
ses clearly showed the presence of this crucial matrix
component in both CT-AM and CT-UC samples, fur-
ther supporting that the cryopreservation process
does not disturb the innate biological components
of the ECM with AM tissue. On the contrary, the
dHACM sample contained neither HC-HA nor
PTX3, only displaying LMW HA and HC1 frag-
ments. While the presence of these smaller proteins
may impart some transient benecial effects to the
dHACM, the lack of the key ECM components HC-
HA and PTX3 suggests reduced longevity of the
active components within the tissue, following the
dehydration process.
While macrophage activation during inamma-
tion is critical to the body’s response to insult, persist-
ence at the site of injury can result in chronic inam-
mation and inability to heal properly, often leading
to tissue damage.52–54 Recent studies have shown the
unique ability of HC-HA/PTX3 to promote the death
of activated macrophages while downregulating pro-
inammatory cytokines and upregulating anti-
inammatory cytokines.25,27,28,30 The results here
demonstrate the ability of CT-AM and CT-UC to sig-
nicantly reduce activated macrophage proliferation
and the substantial ability of CT-UC to enhance acti-
vated macrophage apoptosis when compared to
dHACM. Due to the aforementioned retention of
high levels of HC-HA and PTX3 within the cryopre-
served tissues, the ability of CT-AM and CT-UC to
modulate macrophage cell survival and growth is not
surprising, and is imperative to their success in
wound healing. Similarly, the lack of HC-HA and
PTX3 within dHACM may contribute to the decreased
effectiveness of the dehydrated tissue in reducing
inammation and brosis in vivo.55 Additionally,
cytokines are known to play critical roles in wound
healing.56,57 IL-10 and IL-12 are well studied cytokines
and are known to be anti- and pro-inammatory,
respectively.58–60 Similar to results seen with pure HC-
HA/PTX3,27,28,30 CT-UC signicantly increased IL-10
expression while decreasing IL-12 expression, most
likely due to the substantially higher levels of HC-HA
within CT-UC tissue compared to AM alone.
While the data presented here adequately tests
and describes the differences in structure, func-
tion, and biochemical make-up of both cryopre-
served and dehydrated tissues, the study does have
its limitations. Only a single cryopreservation and
dehydration processing method were analysed and
future experiments including tissues processed by
varying cryopreservation and dehydration proto-
cols should be performed to see if the differences
outlined here extend to other tissues with similar
processing techniques. Additionally, while the
IL-10/IL-12 ratio is indicative of M1 to M2 macro-
phage polarisation, an additional experiments
should include analysis of a larger panel of
cytokines to more fully understand the immu-
nomodulatory actions of the tissue. Finally, to tru-
ly understand the efcacy of the tissue, in vivo
experiments should be performed in various mod-
els of inammation and disease.
Conclusion
Overall, the results of this comparative study bring
to light considerable differences in the structural
and biochemical properties of cryopreserved and
dehydrated foetal tissues. Histological analysis
revealed that while cryopreservation did not dam-
age the delicate architecture of the ECM with the
tissue, dehydration resulted in a compacted AM
morphology and altered the distribution of struc-
tural matrix components. Furthermore, the key
molecules of HC-HA and PTX3 were absent in the
dehydrated tissue and only LMW HA and HC1 pro-
teins were identied, most likely byproducts of
ECM compaction and alteration after preservation.
Not surprisingly, the lack of these critical mole-
cules lessened the biological activity of the dHACM,
as evidenced in the functional assays. Cryopre-
served tissues retained elevated levels of crucial
biological elements and exhibited high functional-
ity in modulating macrophage viability and
cytokine expression. Taken together, this data
strongly suggests that cryopreservation effectively
preserves the structural and biochemical integrity
of AM and UC matrix components, essential for
the anti-inammatory and anti-scarring effects
observed clinically. n
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