Research Article

Cite This: ACS Appl. Mater. Interfaces 2018, 10, 14559−14569 www.acsami.org

Synthetic Nanofiber-Reinforced Amniotic Membrane via Interfacial

Bonding
Huanhuan Liu,†,‡,§,∇ Zhengbing Zhou,†,‡,§,∥,∇ Hui Lin,⊥ Juan Wu,# Brian Ginn,†,‡,§ Ji Suk Choi,†,‡,§
Xuesong Jiang,†,‡,§ Liam Chung,‡,¶ Jennifer H. Elisseeff,‡,§,¶ Samuel Yiu,⊥ and Hai-Quan Mao*,†,‡,§,¶
†
Department of Materials Science and Engineering, and §Institute for NanoBioTechnology, Johns Hopkins University, Baltimore,
Maryland 21218, United States
‡
Translational Tissue Engineering Center, School of Medicine, and ⊥Department of Ophthalmology, Johns Hopkins Wilmer Eye
Institute, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21287, United States
∥
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Department of Hand & Microsurgery, Xiangya Hospital of Central South University, Changsha, Hunan Province 410008, P. R.
China
#
Wuhan Kangchuang Technology, Wuhan, Hubei Province 430073, P. R. China
Downloaded via HACETTEPE UNIV on November 7, 2023 at 13:28:53 (UTC).

¶
Department of Biomedical Engineering, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, United States

ABSTRACT: Severe damage to the ocular surface can result in limbal stem cell (LSC) deficiency, which contributes to loss of
corneal clarity, potential vision loss, chronic pain, photophobia, and keratoplasty failure. Human amniotic membrane (AM) is the
most effective substrate for LSC transplantation to treat patients with LSC deficiency. However, the widespread use of the AM in
the clinic remains a challenge because of the high cost for preserving freshly prepared AM and the weak mechanical strength of
lyophilized AM. Here, we developed a novel composite membrane consisting of an electrospun bioabsorbable polymer fiber
mesh bonded to a decellularized AM (dAM) sheet through interfacial conjugation. This membrane engineering approach
drastically improved the tensile property and toughness of dAM, preserved similar levels of bioactivities as the dAM itself in
supporting LSC attachment, growth, and maintenance, and retained significant anti-inflammatory capacity. These results
demonstrate that the lyophilized nanofiber−dAM composite membrane offers superior mechanical properties for easy handling
and suturing to the dAM, while presenting biochemical cues and basement membrane structure to facilitate LSC transplantation.
This composite membrane exhibits major advantages for clinical applications in treating soft tissue damage and LSC deficiency.
KEYWORDS: amniotic membrane, electrospinning, nanofibers, composite membrane, limbal stem cell transplantation,
macrophage phenotype

1. INTRODUCTION
Limbal stem cells (LSCs) are located in the corneo-scleral
junction, that is, the limbal zone; they are essential for
continuous renewal of corneal epithelium throughout life.1
LSCs may be destroyed or become dysfunctional as a result of
damages to the ocular surface in cases of chemical or thermal
burns or genetic defects, leading to LSC deficiency,2,3 whose
symptoms include chronic inflammation, conjunctivalization,
corneal vascularization, and destruction of the basement
membrane, ultimately resulting in severe vision loss.1−3 LSC
transplantation is a major advance among therapeutic
techniques for reconstruction of the corneal surface, as reported
in a few successful clinical cases using grafts generated from
expanded LSCs to treat patients with LSC deficiency.4,5 LSCs
are typically isolated from the contralateral eye of the patient or
from donor tissues and can be expanded with sufficient quality
and quantity. As the underlining LSC niche is destructed
because of the pathological state of LSC deficiency,2,3 a
functional matrix or scaffold is needed for LSC transplantation
to maximize cell attachment and survival at the repair site.5,6
Human amniotic membrane (AM) remains the most
effective substrate for LSC transplantation to treat patients
with corneal injuries and LSC deficiency.7−9 The efficacy of AM
in promoting healing and regeneration of soft tissue such as
ocular surface reconstruction,10,11 skin transplantation,12−14
and diabetic ulcers treatment15 has been demonstrated in
Received: February 21, 2018
Accepted: April 2, 2018
Published: April 3, 2018

© 2018 American Chemical Society 14559 DOI: 10.1021/acsami.8b03087

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clinical cases and animal studies. One proposed mechanism of
the action for AM-supported transplantation stems from the
low antigenicity and release of various growth factors and
cytokines from the AM, producing biological effects such as
anti-inflammatory, anti-angiogenic, and antifibrotic proper-
ties.16,17 Recent studies have shown that the AM can regulate
inflammatory environments by promoting transformation of
M1 type macrophages to M2 type macrophages and inhibiting
inflammatory response, thus promoting regeneration.18 Anoth-
er important factor is the structure and the extracellular matrix
components in the AM,19 which consists of a single layer of
epithelial cells, a thick basement membrane, and an avascular
stromal matrix. The basement membrane closely resembles
those of the conjunctiva and cornea, especially with regards to
its collagen compositions, strongly facilitates epithelial cell
migration, promotes basal epithelial cell adhesion, and prevents
apoptosis.17,20 These favorable characteristics render the AM an
ideal matrix or scaffold for LSC transplantation.
Even though the bioactivity and structure of AM are best
preserved in freshly prepared samples, the high cost associated
with the preservation and delivery of the fresh AM prevents its
widespread use. On the other hand, it is challenging to process
the AM to a dry form while maintaining its structure, physical
property, and bioactivity. Existing AM processing methods
include wet preservation and lyophilization. Currently available
wet-preserved amniotic products include AmnioGraft, NEOX,
and so forth. The lyophilized AM reduces the cost of storage
and transportation, but it is fragile with poor mechanical
property. Recent efforts to improve the stability, mechanical
properties, and handleability of the AM by using chemical or
enzymatic cross-linkers have only provided limited improve-
ments21−23 with significant loss of bioactive components in the
AM during processing. Other alternative approaches rely on
amniotic extracts or using amniotic extracts in the gel form.24
These approaches retain active substances of the AM to a
certain extent but destroy its natural extracellular matrix
structure, which has been shown to facilitate re-epithelializa-
tion.17,20
Our previous study showed that electrospun polymer
nanofibers offer topographical cues in supporting stem cell
expansion and migration and differentiation.25 For example, an
electrospun poly(ε-caprolactone) (PCL) nanofiber mesh has an
extracellular matrix-mimicking structure with good flexibility
and favorable mechanical properties to support cell adhesion
and growth.26,27 These electrospun polymer fibers can be
surface-functionalized to modulate cell adhesion and present
relevant bioactive moieties.28 We hypothesized that an
electrospun bioabsorbable polymer nanofiber mesh and a
decellularized AM (dAM) sheet can form a composite
membrane by introducing interfacial bonding between the
surface functional groups on electrospun nanofiber mesh and
the protein components in the dAM. The electrospun
nanofiber mesh serves as a reinforcement layer providing
mechanical support to the dAM, while the dAM offers
biochemical cues and basement membrane structure for cell
adhesion, survival, and growth. Here, we describe a method of
preparing a bilayer, bonded electrospun nanofiber−dAM
composite membrane, characterize the improved physical and
functional properties to support rabbit LSC attachment, growth
and maintenance, and report anti-inflammatory capacity of the
composite membrane and its ability to modulate macrophage
phenotype.
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2. MATERIALS AND METHODS
2.1. Preparation of Electrospun PCL, Poly(lactic acid), and
Poly(D,L-lactide-co-glycolide) Nanofiber Meshes. A PCL fiber
mesh was produced by electrospinning of a 12 wt % solution of PCL
(viscosity average MW, 70−90 kDa; Sigma-Aldrich) dissolved in a
mixture of dichloromethane and methanol (4:1, v/v) through a 27-G
needle at an injection speed of 2.5 mL/h to a stationery and grounded
plate collector with a positive potential of 12 kV between the needle
and ground. Fiber deposition onto a stationary and grounded collector
results in random fiber meshes. The thickness of the nanofiber mesh
was controlled to about 40 μm by adjusting the collection time.
Similarly, a poly(lactic acid) (PLA) nanofiber mesh was produced
using a solution of 9 wt % PLA (weight average MW, 78 kDa;
polydispersity index, 1.62; NatureWorks LLC, Minnetonka, MN)
dissolved in a mixture solvent of trichloromethane and dimethylfor-
mamide (9:1, v/v) at a flow rate of 0.5 mL/h using a voltage of 16 kV.
A poly(D,L-lactide-co-glycolide) (PLGA) nanofiber mesh was produced
by electrospinning of a 15 wt % solution of PLGA (50:50, weight
average MW, 30−60 kDa; Sigma-Aldrich) dissolved in 1,1,1,3,3,3-
hexafluoroisopropanol at a flow rate of 0.5 mL/h under a voltage of 6.8
kV.
2.2. Activation of the Carboxyl Group on the Fiber Mesh
Surface. The PCL, PLA, or PLGA fiber meshes were subjected to the
following sequential steps (Figure 1A): (i) treating with plasma in a
Figure 1. (A) Schematic of poly(acrylic acid) (PAAc)-grafting on
electrospun nanofiber mesh, carboxyl group activation and conjugation
of activated fibers with dAM, forming an integrated bilayer composite
membrane. (B) Illustration of mesh compression during interfacial
bonding. PAAc, poly(acrylic acid); NHS, N-hydroxysuccinimide; and
EDC, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide.
radio frequency plasma cleaner (Harrick Plasma) at a medium dose for
10 min, (ii) incubating the treated fiber mesh with 10% acrylic acid
solution containing 0.5 mM NaIO4, followed by exposure to ultraviolet
light at an intensity of 30−50 mW/cm2 for 2 min in an ice bath (0−4
°C), (iii) removing ungrafted PAAc from the surface of the nanofiber
mesh by washing with deionized (DI) water, (iv) measuring the
concentration of the carboxyl groups on fiber mesh by the Toluidine
Blue O (TBO) assay (see below), (v) activating the surface carboxylic
groups by reacting the treated mesh with N-hydroxysuccinimide
(NHS, Sigma) and equal molar of N-(3-dimethylaminopropyl)-N-
ethyl carbodiimide (EDC, Sigma) in 50% ethanol in a glass dish for 4 h
at a molar ratio of 1:4:4 for COOH/NHS/EDC, (vi) removing excess
reagents by thorough rinse with 70% ethanol, and (vii) drying the
modified mesh in a laminar flow hood. The modified nanofiber mesh
was stored at −20 °C and rinsed with sterile phosphate-buffered saline
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(PBS) before use. The surface morphology of the modified mesh was
examined by scanning electron microscopy (SEM) and compared with
that of an unmodified mesh.
2.3. TBO Assay for Surface Carboxyl Group Density on the
Nanofiber Mesh. Modified circular nanofiber mesh, each with an
average surface area of 56.5 mm2 (diameter 6 mm) was incubated in 1
mL of TBO solution (0.5 mM in 0.1 mM NaOH, pH 10) for 5 h at
room temperature under constant agitation. Uncomplexed dye was
removed by washing with NaOH solution (pH 10) and water. The
complexed TBO on PCL mesh was then desorbed from the surface by
incubating the sample in 1 mL of 50% acetic acid (HAc) solution for
10 min under vortexing. TBO concentration in HAc solution was
determined by its optical density measured at 633 nm with a
microplate reader (BioTek Synergy 2). Carboxyl group density on the
surface was calculated from the complexed TBO content assuming
that TBO complexes with the carboxyl groups on the membrane
surface at 1:1 ratio.
2.4. Preparation of Decellularized Human AM. The composite
membrane was prepared using commercially available cryopreserved
human AMs purchased from Bio-Tissue (AmnioGraft, Bio-Tissue
Inc.). After thawing, the human AM sample was spread onto a
nitrocellulose membrane leaving the epithelial cell layer exposed.
Dispase (Millipore) was dissolved in Dulbecco’s modified Eagle’s
medium (DMEM)/F12 medium (Life Technology) to form a 2.5%
(w/v) solution. The AM with nitrocellulose membrane was put into
2.5% dispase solution at 4 °C for 5 h and then washed three times with
sterile PBS. The epithelial cell layer was then removed using an Iris
spatula (Fine Science Tools) under a stereoscope (Nikon SMZ800).
Finally, the dAM was rinsed with sterile PBS for three times and ready
for use.29 To confirm the removal of epithelial cells, the amount of
DNA was quantified by using a DNA isolation kit (DNeasy Blood &
Tissue Kit, QIAGEN). Each test was performed on five different
experimental samples.
2.5. Conjugation of Fiber Mesh to dAM to Form a
Composite Membrane. The process of conjugation of fiber mesh
to the dAM to form a composite membrane is shown in Figure 1A.
The dAM was spread onto a sheet of polytetrafluoroethene (PTFE)
with the epithelial side adjacent to the PTFE. The surface-activated
nanofiber mesh was then spread onto the stromal side of the dAM, and
a second PTFE sheet was placed against the nanofiber mesh. These
assembled layers were placed between two steel plates (Figure 1B) and
mechanical compression was applied for 6 h at 4 °C to reduce the total
thickness to 40 μm, thus ensuring sufficient contact between the
nanofibers and dAM. The dAM and nanofibers were then conjoined
by a condensation reaction of carboxyl groups on the fiber surface and
the amino groups on the protein components in the dAM. After
removing the steel plates and PTFE sheets, the composite membrane
was lyophilized in a freeze dryer (Labconco FreeZone 12L Cascade
Freeze Dry System) and then stored at −20 °C until use.
2.6. Measurement of the Water Content of the Swollen
Composite Membrane. Water content was detected by measuring
the membrane weight before and after swelling in DI water for 10 min.
After being removed from the water, the membranes were hung over a
glass slide for 1 min until no water dripped from them. Membranes
were then removed from the slide and weighed. The water content was
calculated by the following equation:
Water content (%) = [(Ws − Wd)/Ws] × 100%
where Wd is the weight of the dry membrane and Ws is the weight of
the swollen membrane. This testing was performed on five different
experimental samples in each group.
2.7. Mechanical Property Test. The tensile properties of the
dAM (∼10 μm), composite membrane (∼40 μm), and fiber mesh
(∼40 μm) were tested under tension using a Q800 DMA (TA
Instruments). Briefly, membranes were cut into dumb-bell shaped
specimens: the wide ends were 10 mm × 10 mm, and the narrow part
was 1 mm wide by 3 mm long. The ends of the specimen were secured
using metallic grips and then subjected to a force ramp of 0.25 N/min,
starting from 2 mN preload force until specimen failure. The stress−
strain curve was collected for each specimen, and the results for strain
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to failure, toughness, and tensile modulus were calculated. This test
was performed on five different specimens in each group.
Suture retention testing was performed by a modified protocol in a
recent report.30 In brief, each membrane was cut into 2 × 1 cm
samples and placed on the bottom grip of the mechanical testing
machine (BOSE Enduratec ELF 3200). A 7-0 nylon suture was
threaded through the center of the sample, at 2 mm bite depth. The
remainder of the suture was secured to the upper grip, which was
advanced at a crosshead speed of 0.2 mm/s (0.05/s strain rate). The
test ran until either material failure or suture failure occurred or until
the maximum capabilities of the machine were reached. This test was
performed on three different experimental samples in each group.
2.8. Rabbit LSC Isolation and Culture. Fresh eyeballs of New
Zealand white rabbits were purchased from Pel-Freez Biologicals.
Tissues approximately 16 mm in diameter including cornea, limbus,
and conjunctiva were cut from the eyeball using a surgical blade. From
these tissues, 2 mm regions of the limbus were harvested, and 8 mm
diameter portions of central corneas were cut by using a biopsy punch.
The regions of the limbus were incubated with 2.5 mg/mL dispase for
12 h at 4 °C. Under the dissecting microscope, the epithelium sheet
was separated, collected, and digested with 0.25% trypsin/EDTA for 5
min. After blocking with trypsin inhibitor, cells were collected by
centrifugation at 1200 rpm for 5 min and resuspended in LSC culture
medium containing defined keratinocyte-SFM basal medium (In-
vitrogen), defined keratinocyte-SFM growth supplement (Invitrogen),
and 1% Antibiotic-Antimycotic (Gibco).
2.9. Characterization of Cytotoxicity. Cytotoxicities of the
composite, dAM, or nanofiber mesh were assessed according to a
previous report.31 Briefly, samples were placed in a 48-well plate,
covering the bottom of the well, immersed in 200 μL of F12 DMEM-
low glucose medium containing 10% fetal bovine serum (FBS) and
Antibiotic-Antimycotic (Gibco), and incubated for 48 h. The
supernatants were collected as extracts from the scaffolds.
Rabbit LSCs were seeded on a 96-well plate at 103 cells/well for 24
h. The media were replaced with the extracts from the scaffolds
prepared as described above. Low glucose DMEM containing 10%
FBS and Antibiotic-Antimycotic (Gibco) was used as a positive control
and 5% dimethyl sulfoxide as a negative control. After 3 days, the
cytotoxicity was assessed by the alamarBlue assay (Invitrogen).
Following medium removal, the cells were incubated in 10%
alamarBlue cell viability reagent in the media in a 5% CO2 incubator
at 37 °C for 2 h. The absorbance of the culture medium was measured
at 570 nm. Each test group included three different experimental
samples.
2.10. Cell Proliferation Assay. The proliferation of LSCs
cultured directly on the composite, dAM, or nanofiber mesh was
measured by the alamarBlue Assay (Invitrogen). Cells cultured at a
density of 5 × 103 cells/cm2 on different meshes for 1, 3, 5, and 7 days
in LSC culture medium without defined keratinocyte-SFM growth
supplement and then were incubated in 10% alamarBlue cell viability
reagent in the media in a 5% CO2 incubator at 37 °C for 2 h. The
absorbance of the culture medium was measured at 570 nm. This test
was performed on three different experimental samples in each group.
2.11. Isolation and Polarization of Mouse Bone Marrow-
Derived Macrophages. Bone marrow-derived macrophages were
isolated from the femurs of 8 week female C57BL/6 mice, as described
previously.32 Briefly, the femurs of the mice were harvested and the
excess muscle removed. The two ends of the bone were removed using
scissors, and a 21-G needle filled with culture medium was used to
flush the bone marrow cavity. The bone marrow plug suspension was
dispersed by pipetting, filtered through a 70 μm mesh nylon filter, and
centrifuged at 400g for 5 min. Cells were then resuspended in NH4Cl
solution (Stemcell Technology) for 10 min to remove the red blood
cells and centrifuged again at 400g for 5 min. Cells were then
resuspended in complete macrophage culture medium, which is
composed of RPMI 1640 medium (Invitrogen) supplemented with
10% (v/v) FBS(Invitrogen), 1% Antibiotic-Antimycotic (Gibco), and
50 ng/mL recombinant murine M-CSF (Peprotech). Cells were
seeded at 2 × 105 cells/mL into a 6-well cell tissue culture plate and
incubated at 37 °C in 5% CO2. On day 4, culture medium was
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Figure 2. (A) SEM images showing the surface morphology of PCL, PLA, and PLGA fiber meshes before and after modification. Scale bar = 5 μm.
(B) TBO concentrations reflecting the surface densities of carboxyl groups on the PAAc-grafted fiber meshes. (C) Images of the AM and dAM
observed under a bright field microscope (a,b), fluorescence microscope (b,e), and scanning electron microscope (c,f). Cell nuclei were visualized by
DAPI staining under the fluorescence microscope (b,e). Scale bars = 40 μm in (a,b,d,e); 10 μm in (c,f). (D) Detected DNA contents in AM and
dAM samples. AM, amniotic membrane; dAM, decellularized AM.
refreshed with complete medium, and then, the macrophages were
harvested on day 7.
PCL nanofiber−dAM composite membrane, dAM, and PCL
nanofiber mesh were cut into circular samples and fitted into the
wells at the bottom of a 48-well tissue culture polystyrene (TCPS)
plate. The macrophages were reseeded on different substrates in this
plate at a density of 3 × 104 cells per well using bare TCPS wells as a
control and cultured in nonstimulation medium or inflammatory
(classically activated) macrophages (M1) stimulation medium18,33 at
two different stimulation conditions: condition I, 10 ng/mL
lipopolysaccharide (LPS; Sigma) and 5 ng/mL IFNγ (Sigma)
supplemented in the macrophage culture medium; condition II, 100
ng/mL LPS and 50 ng/mL IFNγ in the macrophage culture medium.
The nonstimulated macrophages (M0) cultured in the macrophage
medium were included as a control (condition 0). After culturing for
48 h, gene expression levels of different M1 and M2 makers were
analyzed by real-time PCR as described below.
2.12. RNA Isolation and Real-Time Polymerase Chain
Reaction Analysis. The levels of mRNA for M1 markers (IL1β,
IL6, iNOS, and CD86) and reparative (alternatively activated)
macrophage (M2) markers (Arg1, CD206) of mouse BMDMs
cultured on different substrates were assessed using real-time PCR.
Total cellular RNA was isolated by RNeasy Mini Kits (QIAGEN).
First-strand cDNA synthesis reactions were performed using a High
Capacity cDNA Reverse Transcription Kit (Invitrogen) with a light
cycler apparatus (Biometra TPersonal, German). Real-time PCR was
performed using TaqMan Gene Expression Master Mix (Invitrogen)
with a light cycler apparatus (StepOnePlus Real-Time PCR System,
Applied Biosystems). PCR cycling consisted of 40 cycles of
amplification of the template DNA with primer annealing at 60 °C.
The relative level of expression of each target gene was then calculated
using the 2-ΔΔCt method. The amplification efficiencies of primer
pairs were validated to enable quantitative comparison of gene
expression. All primers were TaqMan primers (Invitrogen). Each
quantitative PCR was performed three times on three different
experimental replicates, and the results were normalized to those
obtained with the reference gene.
2.13. Statistical Analysis. All quantitative data sets are expressed
as mean ± SD. Student’s t-test was performed to assess statistically
significant differences in the results of different experimental groups. A
p value of less than 0.05 is considered statistically significant.
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3. RESULTS
3.1. Modification of PCL, PLA, and PLGA Fiber Mesh
and Surface Characterization. Polyester nanofiber meshes
were prepared from three different polymers by electro-
spinning, and the nanofibers were collected in a random
arrangement with an average thickness of approximately 40 μm.
PCL, PLA, and PLGA fibers were spun at an average diameter
of 517 ± 178, 935 ± 218, and 1260 ± 132 nm, respectively. To
prepare the composite membrane, the electrospun fiber mesh
was grafted with PAAc chains by photoactivated surface grafting
polymerization via free radical initiation (Figure 1). The
carboxyl groups in PAAc chains on the fiber surface were then
activated with NHS/EDC and reacted with protein compo-
nents in dAM using a device we designed for the compression
as shown in Figure 1B, aiming to ensure sufficient contact
between polymer fibers and dAM.
The SEM images did not show any significant morphological
difference between the unmodified and modified nanofiber
mesh (Figure 2A). The TBO test demonstrated that the
carboxyl groups were successfully attached to the surface of the
fiber mesh (Figure 2B). The carboxyl group density on the fiber
surface was calculated from the complexed TBO content,
assuming that TBO complexes with the carboxyl group on the
membrane surface at 1:1 ratio. Therefore, the carboxyl group
densities of PCL, PLA, and PLGA fiber meshes were calculated
as 0.20 ± 0.03, 0.22 ± 0.04, and 0.21 ± 0.02 nmol/mm2,
respectively.
3.2. Decellularization of the Human AM. The epithelial
cell layer of AM was removed by dispase treatment (Figure
2C). The morphology of the epithelial cell layer and cell
nucleus can be seen on the surface of the native AM under a
SEM system and a fluorescence microscope, respectively. After
the decellularization procedure, no cells were visible on the
surface of the dAM (Figure 2C). The total DNA content was
quantified using a DNA isolation kit. Compared to the native
AM (488.2 ± 140.7 ng/mg), the residual amount of DNA in
the obtained dAM (20.7 ± 13.5 ng/mg) was less than 5% of the
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Figure 3. Mechanical properties of composite membrane. (A) General appearance of the composite membranes prepared from different polymer
fibers before lyophilization and after rehydration. (B) Comparison of elastic modulus, strain to failure, ultimate tensile strength, and toughness for
the dAM, unmodified fiber meshes, and composite membranes. (C) Stress−strain curves of the dAM, unmodified fiber meshes, and composite
membranes. (D) Comparison of suture retention strength for the dAM, unmodified fiber meshes, and composite membranes. *p < 0.05, **p < 0.01.

original level (Figure 2D). This is well below the recommended
level for decellularized tissues (<50 ng/mg dry sample).34
3.3. Mechanical Properties of Composite Membrane.
The freshly prepared dAM was difficult to handle; and the
lyophilized dAM was fragile and brittle. In contrast, the
composite membrane was robust and easy to manipulate using
tweezers in both the lyophilized and rehydrated state (Figure
3A). The rehydrated dAM was prone to adhere to itself and
difficult to unfold, whereas the rehydrated composite
membrane prepared from PCL, PLA, or PLGA with the
dAM retained its membrane shape well as a result of higher
levels of stiffness.
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The mechanical properties of the rehydrated dAM,
rehydrated composite membrane, and rehydrated unmodified
fiber mesh were characterized by stress−strain curve analysis
and suture retention tests. As shown in Figure 3B,C, the
rehydrated dAM had moderate stretchability, and its ultimate
tensile strength and toughness were markedly lower than those
of the three composite membranes, confirming that the
composite approach drastically improved the tensile property
and toughness of dAM. Compared to the untreated fiber mesh,
the elastic moduli of the composite membranes were higher,
though the toughness was lower, likely due to the hydration of
the membranes.
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Figure 4. (A) General appearance of dAM, the composite membranes prepared from PCL fiber meshes with different thicknesses before
lyophilization and after the rehydration in comparison with the unmodified and surface treated PCL fiber meshes (40 μm). (B) Water content of
dAM, the PCL fiber−dAM composite membranes with different thicknesses, and the untreated PCL fiber.

Suture retention strength is commonly used as a measure of

the ability of sutures to withhold implants to surrounding
tissue, which is usually performed according to the AAMI/ISO/
ANSI7198 Standard.35,36 Here, we adopted a slightly modified
protocol to compare the ability of our composite membrane
with the dAM in terms of their abilities to tolerate tear force
during suture pullout. As shown in Figure 3D, suture retention
strengths for all three composite membranes preserved
significant portions (50−65%) of their corresponding nanofiber
meshes without any modification. Compared to the dAM
samples, the measured failure forces were 14.7-, 10.5-, and 13.2-
times higher for composite membranes prepared from PCL,
PLA, and PLGA nanofibers, respectively. Considering the need
to balance flexibility and toughness with strength, modulus, and
suture retention ability for the application in LSC trans-
plantation, we selected electrospun PCL fiber−dAM composite
membrane for the following studies.
PCL fiber−dAM composite membranes prepared with three
different thicknesses 40, 80, and 120 μm were further
characterized for their mechanical property in dry and
rehydrated states (Figure 4). The composite membrane with
greater thickness was more easily manipulated in the rehydrated
state; nonetheless, the water absorption degrees were similar
among the dAM, composite membranes, and surface-modified
fiber meshes. The unmodified fiber mesh however was too
hydrophobic to imbibe water. For all following studies, we have
used the composite membranes prepared from 40 μm thick
PCL nanofiber meshes.
3.4. Surface and Cross-Sectional Features of the
Composite Membrane. The mechanical compression
improved contact between the dAM and the PCL fiber mesh,
Figure 5. (A) Visual appearance of the lyophilized composite
thus facilitating the conjugation reaction between the fiber
membranes prepared using the same conditions with/without the
surface and the dAM components. The PCL fiber−dAM compression step during conjugation or with/without the fiber surface
composite membrane formed stronger bonding between the functionalization step, confirming that both steps are required to
two layers of materials (Figure 5A). On the other hand, lack of generate an integrated composite membrane. (B) SEM images
compression or activation of the fiber surface resulted in poor showing surface morphology at the fiber−dAM boundary (a,b,e) and
binding between dAM and PCL fiber mesh, as it was easy to center (c,d,f) sections of the composite membrane with (b,c,e,f) and
delaminate the two layers after lyophilization. without (a,d) mechanical compression. Red arrows indicate the
The surface morphology of the dAM−PCL composite interface between the dAM and the fiber mesh. Scale bars, 10 μm. (C)
Cross-sectional views of dAM (a) and the composite membrane (b,c)
membrane with and without compression was observed by following H&E staining (a−c) and under SEM (d−f, composite
SEM (Figure 5B). Without compression, the conjugation and membrane). Scale bars, 20 μm in (a−c); 10 μm in (d); 2 μm in (e);
binding between the fiber mesh and dAM was not uniform, and 1 μm in (f).
with some regions of the dAM detaching from the fiber mesh
after lyophilization. Following the compression step, the The cross sections of the dAM and PCL fiber−dAM
conjugation was more thorough, and the electrospun fiber composite membrane were examined under the optical
features were visible from the dAM side of the membrane as a microscope following H&E staining and by SEM following
result of imprinting (Figure 5B-b,c,e,f). lyophilization (Figure 5C). As a control, H&E-stained AM
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Figure 6. (A) Fluorescence images showing cell morphology of LSCs cultured on different substrates without including the defined keratinocyte-
SFM growth supplements in the medium. Scale bars, 50 μm. (B) Relative cell proliferation rate of LSCs cultured on various substrates in comparison
with the TCPS plate under the same culture condition as indicated in (A). (C) Relative viability of LSCs cultured with medium extracts from a 2 day
culture of dAM, PCL fibers, composite membrane, and TCPS in comparison with a medium control.

Figure 7. (A) Relative expression levels of LSCs markers, ABCG2, P63, and K14, in rabbit LSCs cultured on dAM, unmodified nanofibers, and
composite membrane in comparison with TCPS. Primary rabbit LSCs were cultured on various substrates for 5 days in serum-free medium without
the defined keratinocyte-SFM growth supplement. (B) P63 expression profiles in LSCs following a 5 day culture on dAM, unmodified nanofibers,
composite membrane, and TCPS. Scale bar = 50 μm. (C) Percentage of P63-positive cells on day 5 among LSCs cultured on dAM, unmodified
nanofibers, composite membrane, and TCPS. *p < 0.05, **p < 0.01.

showed a single epithelial layer on one side and a sponge-like
stromal layer on the other side (Figure 5C-a). The cross section
of the PCL−dAM composite membrane revealed an electro-
spun fiber partially merging into the stromal side of the dAM
(Figure 5C-b,c), and SEM analysis of the ultrastructure of the
composite membrane cross section reflecting a tight connection
between the dAM and the PCL fiber mesh (Figure 5C-d−f).
3.5. Morphology and Proliferation Rate of Rabbit
LSCs Cultured on the Composite Membrane. To compare
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the bioactivity of the dAM and that of the composite
membrane, primary rabbit LSCs were cultured in serum-free
medium without the defined keratinocyte-SFM growth supple-
ment. Figure 6A shows that the LSCs on the dAM, composite
membrane, and TCPS spread well in a spindle shape; on the
other hand, cells on the PCL fiber mesh appeared smaller.
Scaffold toxicity analysis did not show any significant influence
to LSCs by all scaffold material extracts (Figure 6C). In
addition, the proliferation rate of LSCs cultured on dAM,
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Figure 8. Expression levels of M1 (A) and M2 (B) markers in mouse bone marrow-derived macrophages cultured on dAM, unmodified fiber
meshes, and composite membranes, in comparison with cells cultured on the TCPS substrate for 48 h. Macrophages were cultured in absence of any
additional stimulation (condition 0), lower (10 ng/mL LPS and 5 ng/mL IFNγ, condition I), or higher (100 ng/mL LPS and 50 ng/mL IFNγ,
condition II) concentrations of M1 polarization cytokines. Each bar represents mean ± SD (n = 3). *p < 0.05, **p < 0.01, &p < 0.05, &&p < 0.01, #p
< 0.05, ##p < 0.01.

unmodified PCL fiber mesh, or composite membrane was
quantified by the alamarBlue assay (Figure 6B). LSC
proliferation on the TCPS dish was included as a positive
control, which exhibited a moderate increase until day 3 but
decreased from day three through day 7. Cells on the PCL
fibers followed a similar trend by increasing a little from days 1
to 3 but started to decrease on day 5. Importantly, the
proliferation of LSCs on the dAM increased from day 1 to day
7. Similar trends were observed in PCL fiber−dAM composite
membrane. On the basis of these findings, we speculate that
LSCs could maintain viability for 7 days with the nutrient
secreting from the dAM and the PCL composite membrane.
3.6. Effect of the Substrate on LSC Marker Gene
Expression. Following a 5 day culture of primary rabbit LSCs
on dAM, PCL fiber mesh, and composite membrane in serum-
free medium without defined keratinocyte-SFM growth supple-
ment, the expression levels of LSCs markers, ABCG2, P63, and
K14, increased significantly in cells on the dAM and the PCL
fiber−dAM composite membrane in comparison with cells on
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the TCPS substrate (Figure 7A): ABCG2 expression was 3.4-
fold (p < 0.01) and 5.0-fold (p < 0.05) higher, P63 expression
was 2.4-fold (p < 0.05) and 2.3-fold (p < 0.01) higher, and K14
expression was 2.6-fold (p < 0.01) and 2.5-fold (p < 0.05)
higher on the dAM and PCL fiber−dAM composite membrane,
respectively, compared to TCPS. However, there were no
significant differences between dAM and PCL fiber−dAM
composite membrane in the relative expression levels of all
three markers. To confirm these results, the immunofluor-
escence assay of P63 was performed. Consistent with the real-
time Q-PCR results, the percentages of P63-positive cells were
significantly higher on both dAM and PCL fiber−dAM
composite membrane compared with TCPS control (Figure
7B,C). These results indicate that the PCL fiber−dAM
composite membrane retained the essential bioactive cues of
the dAM in promoting LSC maintenance.
3.7. Effect of the Composite Membrane on Macro-
phage Polarization. Macrophages are the first responder
when biomaterials are first introduced into the body, and their
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responses to the materials trigger a cascade of complex immune
response, so it is important to evaluate their phenotypic
changes with our materials. To evaluate the anti-inflammatory
property of the PCL fiber−dAM composite membrane,
phenotype transition from pro-inflammatory M1 to pro-
regenerative M2 for macrophages cultured on the composite
was characterized in comparison with those on dAM, PCL fiber
mesh, or TCPS. Mouse bone marrow-derived macrophages
(M0) were cultured on these substrates and then subjected to
M1 polarization condition in the presence of different
concentrations of LPS and IFNγ. After culturing for 48 h,
gene expression levels of different M1 and M2 makers were
analyzed by real-time Q-PCR. As shown in Figure 8, without
M1 stimulation (no LPS or IFNγ), M1 and M2 marker
expression levels in macrophages cultured on all four substrates
were similar. Following stimulation with LPS and IFNγ,
expression profiles of the pro-inflammatory (M1) markers,
IL1β, IL6, iNOS, and CD86, in macrophages cultured on TCPS
increased proportionally with the concentration of the
stimulation cytokines; the expression level of M2 marker
CD206 decreased accordingly.
In the presence of lower concentrations of LPS (10 ng/mL)
and IFNγ (5 ng/mL), compared to the cells on TCPS,
expression levels of most M1 markers tested were similar
among these four substrates (Figure 7A). However, the iNOS
expression levels were 1.7-fold (p < 0.05) and 5.4-fold (p <
0.01) lower on the dAM and the PCL fiber−dAM composite
membrane, respectively, compared with the TCPS control. The
IL6 expression level was 2.8-fold lower (p < 0.05) in
macrophages on the dAM than that on the TCPS.
When compared against TCPS control in the presence of
higher concentrations of LPS (100 ng/mL) and IFNγ (50 ng/
mL), expression levels of most M1 markers tested in cells
cultured on dAM and the composite membrane were down-
regulated. IL1β expression levels in macrophages on the dAM
and the composite membrane were 3.9-fold (p < 0.01) and 1.7-
fold (p < 0.05) lower than those on the TCPS. IL6 expression
was 3.1-fold (p < 0.05) lower on the composite membrane than
that on the TCPS. The levels of iNOS were 1.5-fold (p < 0.05)
and 3.6-fold (p < 0.01) lower, and CD86 were 1.6-fold (p <
0.05) and 2.0-fold (p < 0.01) lower, on dAM and the composite
membrane, respectively, than those on the TCPS control
(Figure 8A).
In both stimulation conditions, M2 markers Arg1 and
CD206 up-regulated significantly on the composite membrane
in comparison with TCPS. In the presence of the lower
concentration of LPS (10 ng/mL) and IFNγ (5 ng/mL)
[condition I], Arg1 expressions on dAM and the composite
membrane were 13.5-fold (p < 0.01) and 4.1-fold (p < 0.01)
higher, respectively, than that in TCPS control; and CD206
expression levels were 78.5-fold (p < 0.01) and 174.5-fold (p <
0.01) higher, respectively, than that in cells on TCPS (Figure
7B). Under the stimulation condition II with higher
concentrations of LPS (100 ng/mL) and IFNγ (50 ng/mL),
Arg1 levels were 1.3-fold and 3.5-fold (p < 0.05) higher on
dAM and the composite membrane, respectively, than that on
TCPS; CD206 expression levels were 4573-fold (p < 0.01) and
12 109-fold (p < 0.01) higher on dAM and the composite
membrane, respectively, compared with that in cells cultured on
TCPS (Figure 8B).
Taken together, under the inflammatory stimulation
condition, both dAM and PCL fiber−dAM composite
membrane significantly reduced pro-inflammatory marker
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Research Article
(M1) expression and enhanced the expression of reparatory
marker (M2) expression, in comparison with TCPS control.
This suggests that the composite membrane has anti-
inflammatory properties similar to that of dAM and may
promote the polarization of some macrophages into M2 type,
particularly under stronger inflammatory cues.
When compared dAM to the PCL fiber−dAM composite
membrane under inflammatory conditions, there were no
significant differences in IL1β and CD86 expression but
significant differences in iNOS, IL6, Arg1, and CD206
expression. The iNOS expression levels were 3.1-fold (p <
0.05) and 2.4-fold (p < 0.05) lower on the composite
membrane than those on dAM under the low and high
stimulation conditions, respectively. IL6 expression was 4.6-fold
(p < 0.05) higher on the composite membrane than on dAM
under the low stimulation condition but 3.0-fold (p < 0.05)
lower on the composite membrane than that on dAM under
the high stimulation condition.
Arg1 expression level was 3.3-fold (p < 0.01) higher on the
composite membrane than that on dAM under the low
stimulation condition and 2.6-fold (p < 0.05) lower on the
composite membrane than on dAM under the high stimulation
condition. Similarly, CD206 expression levels were 2.2-fold (p
< 0.05) and 2.6-fold (p < 0.01) higher on the composite
membrane than on dAM under the low and high stimulation
conditions, respectively.
4. DISCUSSION
Despite the excellent regenerative capacity afforded by the AM,
the high preservation cost and relatively weak mechanical
strength have significantly limited the application of the fresh
and dehydrated AM. In this present study, a novel electrospun
fiber−dAM composite membrane has been designed as a dAM
substitute for LSC transplantation and soft tissue repair. The
electrospun fiber−dAM composite membrane combined the
advantage of dAM and electrospun fiber mesh. Because of the
facile conjugation reaction and sequential reaction steps, that is,
activation of the fiber surface and removing excess reagents
before reacting with dAM under compression, the obtained
composite membrane retained nearly full bioactivity of the
dAM.
Many methods have been applied to enhance AM
mechanical properties and handleability by treatment with
carbodiimide (e.g. EDC/NHS22 or EDC with L-lysine23) and
enzymatic (e.g. transglutaminase21) cross-linkers, although
these methods only provided limited improvement in
mechanical properties and these cross-linking process involves
random self-cross-linking of the dAM, leading to potential loss
of bioactivity and reduction of biocompatibility. Alternatively, a
hydrogel form was developed using dAM extracts to facilitate
the usage;24 however, this hydrogel form loss the membrane
structure and physical support for LSCs. In our study, the
electrospun fiber mesh was pretreated first with EDC cross-
linker in the presence of NHS for activation. Following
removing of excess reagents by washing, these active ester
groups were then reacted with dAM. This method did not
expose dAM with EDC directly, thus avoiding self-cross-linking
reactions occur among the ECM molecules within the dAM.
Therefore, the composite membrane retained maximal
bioactivity of the dAM, as it showed similar capacity as the
dAM in supporting rabbit LSC attachment, proliferation, and
phenotype maintenance under the expansion condition. More
importantly, the composite membrane, dAM, and to a large
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extend the electrospun fiber mesh significantly promoted M2
polarization of the primary mouse macrophages cultured under
the inflammatory activation condition in vitro, while moder-
ately reducing their M1 polarization. The bioactivity and
efficacy of the composite membrane in supporting LSC
transplantation remained to be tested in future in vivo
experiments.
The interfacial bonding between the electrospun fiber surface
and the dAM protein components generates an integrated
membrane structure that not only drastically improved the
mechanical properties of the dAM but also rendered the
electrospun fiber mesh more elastic. In addition, the composite
membrane also retained excellent suture retention ability of the
electrospun fiber mesh.
The stronger elasticity and ductility of the membrane make it
easier to be curved and sutured to conform to the wound
surface. Previous research demonstrated the success of AM
application for the regeneration of peripheral nerves.37
However, it is hard to curve the wet AM into a tube or wrap
it around the injured nerve during an operation. The composite
membrane is relatively easy to wrap into a tube or around a
nerve, both in wet or dry state, which is promising for nerve
repair applications. Other parameters such as the alignment and
diameter of fibers in the composite could be easily varied as
needed.
The ECM microenvironment is crucial to cell morphology,
adhesion, expansion, and differentiation.38 Similar to the effects
promoted by different biochemical cues, a number of
biomimetic scaffolds have been developed with specific physical
properties and topographical cues to influence cell behavior.39
It has been demonstrated by several studies that longitudinally
aligned nanofibers increase the efficiency of neuronal differ-
entiation of adult neural stem cells in vitro and promote
peripheral nerve regeneration in vivo.25,40,41 Similarly, several
studies showed that the nanofiber mesh promotes teno-lineage
differentiation of mesenchymal stem cells and human induced
pluripotent stem cells42,43 and phenotype maintenance.28,44 It
has also been demonstrated that the dAM can significantly
reduce the amount of postsurgery adhesion.37,45 Therefore, the
composite membrane prepared from a nanofiber mesh may
represent a promising biomaterial for use in bioengineered
peripheral nerve and tendon repair.
Because of these significant improvements in physical
property and bioactivity, the composite membrane promises a
broad range of applications in treating soft tissues injures and
diseases besides LSC deficiency. In addition to the tested PCL
fiber−dAM composite, other parameters including the number
of layers, broad range of shapes, and the polymer materials used
to produce fibers are all tunable. Additional layers of the dAM
could be applied to the composite to add other biological
activities and functions.46,47
The excellent lyophilization and rehydration properties of the
composite membrane make it easy to store on the shelf and
easy to transport. In comparison with both the freshly prepared
and lyophilized dAM, the composite membrane substantially
reduces the storage and transportation costs, besides improving
the mechanical properties and maintaining the bioactivity and
biocompatibility. All of these favorable features enable the
clinical translation of the new nanofiber−dAM hydrogel
composite membrane.
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Research Article
■ AUTHOR INFORMATION
Corresponding Author
*E-mail: hmao@jhu.edu.
ORCID
Hai-Quan Mao: 0000-0002-4262-9988
Author Contributions
∇
H.L. and Z.Z. contributed equally to this article.
Notes
The authors declare the following competing financial
interest(s): Dr. Juan Wu (a co-author) is an employee of
Wuhan Kangchuang Technology Limited that funded this
study.
■ ACKNOWLEDGMENTS
This work was supported by Wuhan Kangchuang Technology
Co. Ltd. and partially supported by a grant (DMR1410240)
from the National Science Foundation.
■
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