Biodegradable photo-crosslinked alginate nanofibre scaffolds with tuneable physical properties, cell adhesivity and growth factor release

Abstract

Nanofibrous scaffolds are of interest in tissue engineering due to their high surface area to volume ratio, interconnected pores, and architectural similarity to the native extracellular matrix. Our laboratory recently developed a biodegradable, photo-crosslinkable alginate biopolymer. Here, we show the capacity of the material to be electrospun into a nanofibrous matrix, and the ability to enhance cell adhesion and proliferation on these matrices by covalent modification with cell adhesion peptides. Additionally, the potential of covalently incorporating heparin into the hydrogels during the photopolymerisation process to sustain the release of a heparin binding growth factor via affinity interactions was demonstrated. Electrospun photo-crosslinkable alginate nanofibrous scaffolds endowed with cell adhesion ligands and controlled delivery of growth factors may allow for improved regulation of cell behaviour for regenerative medicine.

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SI Jeong et al. Biodegradable alginate nanobre scaffolds
European Cells and Materials Vol. 24 2012 (pages 331-343) ISSN 1473-2262
Abstract
Nanobrous scaffolds are of interest in tissue engineering
due to their high surface area to volume ratio, interconnected
pores, and architectural similarity to the native extracellular
matrix. Our laboratory recently developed a biodegradable,
photo-crosslinkable alginate biopolymer. Here, we
show the capacity of the material to be electrospun into
a nanofibrous matrix, and the ability to enhance cell
adhesion and proliferation on these matrices by covalent
modication with cell adhesion peptides. Additionally,
the potential of covalently incorporating heparin into
the hydrogels during the photopolymerisation process to
sustain the release of a heparin binding growth factor via
afnity interactions was demonstrated. Electrospun photo-
crosslinkable alginate nanobrous scaffolds endowed with
cell adhesion ligands and controlled delivery of growth
factors may allow for improved regulation of cell behaviour
for regenerative medicine.
Keywords: Alginate; electrospinning; photopolymerisation;
biomaterials; heparin; tissue engineering.
*Address for correspondence:
Eben Alsberg
Department of Biomedical Engineering and Orthopaedic
Surgery
Case Western Reserve University
Wickenden Building, Room 204
10900 Euclid Avenue
Cleveland, OH 44106, USA
Telephone Number: +1 216 368 6425
FAX Number: +1 216 368 4969
E-mail: eben.alsberg@case.edu
Introduction
Wounds occur when the skin is damaged; this can be
caused by trauma, burns, diabetic ulcers and surgical
procedures. Wound healing is a complex series of events
that involves the responses of cells, growth factors and
cytokines as well as the extracellular matrix (ECM) (Janis
et al., 2010). Conditions such as poor circulation, other
illnesses and age can cause wound healing to be delayed
or impaired, resulting in chronic non-healing wounds (Wu
et al., 2010). Some tissue engineering strategies for wound
repair, such as that presented here, seek to provide an
articial dermal layer comprised of a biomaterial scaffold
into which dermal broblasts can migrate and proliferate.
Generally, biomaterials for wound repair should be non-
toxic, exible, durable and non-antigenic during their
contact with the tissue.
Additionally, it may be benecial to deliver bioactive
factors from the biomaterial scaffolds to further enhance
the tissue regeneration. Several growth factors have
been shown to be important in wound healing, including
epidermal growth factor, platelet-derived growth factor,
transforming growth factor beta and broblast growth
factor-2 (FGF-2) (Barrientos et al., 2008). FGF-2 is
important for cell proliferation and broblast inltration
into the wound (Barrientos et al., 2008), and chronic
wounds have been found to have decreased levels of FGF-2
(Robson, 1997). Thus, delivering FGF-2 to a wound from
a biomaterial serving as temporary dermal matrix may
aid in the healing process. This has been examined using
FGF-2-laden gelatin microparticles in collagen scaffolds
(Park et al., 2009), FGF-2-laden poly(lactic-co-glycolic)
acid (PLGA) microparticles in alginate scaffolds (Perets
et al., 2003), FGF-2 encapsulated in collagen-heparin
hydrogels (Nillesen et al., 2007), poly(ethylene glycol)
(PEG) hydrogels (Andreopoulos and Persaud, 2006),
heparin-PEG hydrogels (Benoit and Anseth, 2005),
polyelectrolyte multi-lms comprised of poly(beta-amino
esters) and chondroitin sulphate or heparin (Macdonald et
al., 2010), silk broin scaffolds (Wongpanit et al., 2010),
poly(ether)urethane-polydimethylsiloxane and fibrin
composite hydrogels (Briganti et al., 2010), chitosan/
hydroxyapatite scaffolds (Tigli et al., 2009), chitosan-
alginate polyelectrolyte scaffolds (Ho et al., 2009),
sulphated alginate hydrogels (Freeman et al., 2008) and
PLGA electrospun nanobres (Sahoo et al., 2010).
Electrospun nanobres offer an architecture which
is a promising option for wound healing matrices; their
nanoporous nature may aid in the transport of oxygen
to the wound while keeping bacteria out, and bioactive
BIODEGRADABLE PHOTO-CROSSLINKED ALGINATE NANOFIBRE SCAFFOLDS
WITH TUNEABLE PHYSICAL PROPERTIES, CELL ADHESIVITY
AND GROWTH FACTOR RELEASE
Sung In Jeong1,§, Oju Jeon1, Melissa D. Krebs1, Michael C. Hill1 and Eben Alsberg1,2,*
1Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA
2Department of Orthopaedic Surgery, Case Western Reserve University, Cleveland, OH, USA
§Current address: Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute,
1266 Sinjeong-dong, Jeongeup-si Jeollabuk-do, 580-185, Republic of Korea

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SI Jeong et al. Biodegradable alginate nanobre scaffolds
factors can be incorporated into the nanobres to further
promote healing (Zhong et al., 2010). Furthermore, the
nanobrous structure of electrospun scaffolds mimics
the structure of the ECM in which cells naturally reside,
and has been shown to regulate many cellular processes
such as adhesion, spreading, proliferation, alignment and
differentiation (Murugan and Ramakrishna, 2006; Pham
et al., 2006). In addition, the high surface area to volume
ratio maximises cell interactions with these materials, and
the subsequent potential for material mediated signalling.
Natural materials such as collagen (Powell et al., 2008),
chitin (Noh et al., 2006) and gelatin (Powell and Boyce,
2008), as well as synthetic materials such as PEG (Casper
et al., 2005) have been examined for use as electrospun
scaffolds in wound healing. The use of hydrophilic
biomaterials as electrospun nanobres may be benecial in
retaining an appropriate balance of moisture at the wound
to aid in the healing process (Zahedi et al., 2010).
Alginate, a hydrophilic biocompatible polymer
derived from seaweed, has been electrospun and ionically
crosslinked with calcium to form nanobrous scaffolds;
the cell adhesivity of these scaffolds can be regulated
by modifying the alginate backbone with cell adhesive
peptides found in natural ECM molecules (Jeong et al.,
2010). However, it can be difcult to control the physical
properties of ionically crosslinked alginate. Furthermore,
although bioactive factors can be incorporated into the
alginate bres, they will rapidly diffuse from the water-
swollen network. Our group has recently engineered
photo-crosslinkable methacrylated alginate, which
provides the capacity to control its physical properties
such as degradation rate, swelling, and mechanical
properties (Jeon et al., 2009) by varying the degree of
alginate methacrylation. Furthermore, the methacrylated
alginate, which is initially non-adhesive to cells, can also be
covalently modied with cell adhesive peptides to regulate
cell behaviours such as cell attachment, spreading and
proliferation on or within the matrices (Jeon et al., 2010)
and heparin to control and sustain the release of heparin-
binding growth factors (Jeon et al., 2011). While many
biomaterials have been electrospun to permit investigation
of nanobre structure on cell behaviour, few are at the same
time biodegradable with the capacity for both controlled
cell adhesion and bioactive factor delivery. Here we report
on the ability to electrospin the methacrylated alginate
into nanobres and crosslink the bres using ultraviolet
(UV) light to form stable nanofibrous scaffolds. The
alginate polymer backbone can be covalently modied
with cell-adhesive peptides to control cell adhesion (Jeon
et al., 2010) (Fig. 1a). Methacrylated heparin (Fig. 1b)
can be blended with methacrylated alginate so that upon
crosslinking, the alginate scaffold will contain covalently
linked heparin to mediate the sustained release of
incorporated growth factors (Jeon et al., 2011). Both the
peptides and the heparin remain bioactive following the
electrospinning process. The resultant nanobres thus have
tuneable physical properties, cell adhesive properties and
growth factor release proles. These electrospun alginate
scaffolds have much promise for wound healing and other
regenerative medicine applications.
Materials and Methods
Synthesis of methacrylated alginate, RGD-modied
methacrylated alginate and methacrylated heparin
Low molecular weight sodium alginate (37,000 g/mol) was
prepared by irradiating Protanal LF 20/40 (196,000 g/mol,
FMC Biopolymer, Philadelphia, PA, USA) at a gamma
dose of 5 Mrad. Unmodified methacrylated alginate
(UMA) and RGD-modied methacrylated alginate (RMA)
were synthesised as described previously, at a theoretical
methacrylation of 45 % (25 % actual) (Jeon et al., 2009;
Jeon et al., 2010). The methacrylated heparin was prepared
as previously described, at a theoretical methacrylation
of two carboxylic acid groups per heparin molecule (~1.4
actual) (Jeon et al., 2011).
Preparation of methacrylated alginate-PEO
nanobrous scaffolds and photo-crosslinking
UMA and RMA were dissolved in ultrapure deionised water
(diH2O) with 0.05 % (w/v) photoinitiator (Irgacure D-2959,
Sigma-Aldrich, St. Louis, MO, USA) at concentrations
from 1.0 to 8.0 % (w/v). Methacrylated heparin was
dissolved in this RMA solution at a concentration of 1.0 %
(w/v) (HRMA). Poly(ethylene oxide) (PEO, 900 kDa,
Sigma-Aldrich) was dissolved in diH2O with 0.05 % (w/v)
photoinitiator at a concentration of 4.0 % (w/v). The UMA,
RMA, and HRMA were mixed with the PEO at a blending
ratio of 50:50 alginate:PEO for 1 day at room temperature
using a rotating hybridisation incubator (Model 400;
Robbins Scientic, Sunnyvale, CA, USA).
Fig. 1. Chemical structures of (a) methacrylated alginate modied with RGD-containing peptide and (b) methacrylated
heparin.

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SI Jeong et al. Biodegradable alginate nanobre scaffolds
For electrospinning, the blended solutions were loaded
in a 5 mL plastic syringe (Henke Sass Wolf, Tuttlingen,
Germany), tted with a stainless-steel blunt-ended needle
(20G, NanoNC, Seoul, Korea). The plastic syringe was
placed in an infusion pump (Model 22; Harvard Apparatus
Inc., Holliston, MA, USA) and the stainless-steel needle
was connected to the positive output of a high-voltage
power supply (AU 60PO; Matsusada, Kusatsu, Japan). A
custom-made rotating collecting drum (outer diameter:
100 mm, length: 250 mm; NanoNC) was wrapped with
aluminium foil and positioned at a fixed distance of
150 mm from the needle. The applied voltage and ow rate
of the infusion pump were xed to 10.4 kV and 0.01 mL/
min, respectively, and the total spinning time for these
scaffolds was 3 h. A list of the solutions examined for
electrospinning is presented in Table 1.
After fabrication, all nanobres were photo-crosslinked
with 365 nm UV light (Model B-100AP, UVP LLC,
Upland, CA, USA) at <1 mW/cm2 at a distance of 100 mm
from the light for 10 min and dried overnight at room
temperature. The crosslinked nanobres were then punched
into disks (diameter 30 mm) using a punch (McMaster
Carr, Elmhurst, IL, USA). To extract the PEO from the
photo-crosslinked scaffolds, they were incubated in 5 mL
of diH2O or Dulbecco’s Modied Eagle Medium (DMEM)
in 6-well plates at 37 °C for 5 days with slow shaking.
The diH2O and DMEM solutions were changed every day.
The PEO-extracted scaffolds were rinsed three times with
diH2O, frozen at -80 °C overnight, and lyophilised for 3
days.
Morphologies of electrospun photo-crosslinked
alginate scaffolds
The morphologies of the photo-crosslinked alginate
nanobres before and after PEO extraction were examined
using a scanning electron microscope (SEM, S-4500,
Hitachi, Tokyo, Japan). The samples were coated with
gold using a sputter-coater (E-1030, Hitachi) and scanned
at an acceleration voltage of 5 kV. One representative
photomicrograph from each sample was used to measure
the diameters of 50 bres using image analysis software
(Image-Pro Plus 6.0, Bethesda, MD, USA).
Mechanical properties of the nanobrous scaffolds
The tensile strength, Young’s moduli, and elongation
at break of the photo-crosslinked MA/PEO nanobres
(UMA84, RMA84, and HRMA84) were determined
by performing constant strain rate tensile tests using a
Rheometrics Solids Analyser (RSAII, Rheometrics Inc.,
Piscataway, NJ, USA) equipped with a 10-N load cell. The
photo-crosslinked MA/PEO nanobres were prepared as
described above and individual scaffolds were cut from
the electrospun mat with dimensions of 10×5 mm2 (n = 5)
and attached to cardboard using epoxy resin. The sample
was centred in a 5 mm slot in the centre of the cardboard
and then glued to standardise the gauge length (Fig. 2).
After measuring the sample thickness using a pair of
callipers, the cardboard was loaded into the clamps of the
Rheometrics device and cut as indicated in Fig. 2. Tensile
tests were performed on the scaffolds at room temperature
at a cross-head speed of 0.6 mm/min. The tensile moduli
Table 1. Solution blends used for electrospinning of methacrylated alginate/PEO.
Sample code
Unmodied and
RGD-modied
methacrylated
alginate (wt %)
PEO
comcentration
(wt %)
Alginate: PEO
vol % : vol %
(nal wt % : wt %)
UMA14 1.0 4.0 50 : 50 (0.5 : 2.0)
UMA24 2.0 4.0 50 : 50 (1.0 : 2.0)
UMA44 4.0 4.0 50 : 50 (2.0 : 2.0)
UMA84 8.0 4.0 50 : 50 (4.0 : 2.0)
RMA84 8.0 4.0 50 : 50 (4.0 : 2.0)
HRMA84 8.0 4.0 50 : 50 (4.0 : 2.0)
Fig. 2. Schematic illustration
of the sample holder used for
uniaxial tensile strength testing
of electrospun mats. The resulting
gauge length was 5 mm.

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SI Jeong et al. Biodegradable alginate nanobre scaffolds
of the electrospun nanobrous scaffolds were determined
from the slope of stress vs. strain plots, limited to the rst
linear 2 % strain of the plots.
ATR-FTIR of the nanobrous scaffolds
To examine the chemical composition of the photo-
crosslinked UMA84, RMA84 and HRMA84 nanobres,
attenuated total reectance-Fourier transform infrared
(ATR-FTIR) spectroscopy (Excalibur FTS 3000, Bio-Rad/
Digilab; Bio-Rad, Hercules, CA, USA) was performed.
ATR spectra were recorded at 64 scans with a resolution of
40 cm-1 and a scanning range between 2000 and 600 cm-1.
Degradation of the scaffolds
The degradation of the photo-crosslinked alginate
nanofibrous scaffolds was investigated. The photo-
crosslinked, PEO-extracted, lyophilised UMA84,
RMA84 and HRMA84 nanobrous scaffolds (diameter
20 mm, n = 2 per time point) were placed in closed
50 mL polypropylene conical tubes containing 30 mL
of Dulbecco’s Modied Eagle’s Medium (DMEM) for 3
weeks at 37 °C. The DMEM was changed every 3 days.
Samples were then washed with diH2O and lyophilised. At
1, 2, and 3 weeks the samples were imaged using SEM.
Interaction of cells with the photo-crosslinked
alginate nanobres
Primary human dermal fibroblasts (HDFs, ATCC,
Manassas, VA, USA) were maintained in DMEM
containing 4.5 g/L glucose (DMEM-HG, HyClone,
Logan, UT, USA) supplemented with 10 % fetal bovine
serum (FBS, HyClone) and 1 % penicillin / streptomycin
(P/S, HyClone) at 37 °C with 5 % CO2 in a humidied
environment. Cells were used at passages 4-5.
Photo-crosslinked UMA84, RMA84, and HRMA84
PEO-extracted scaffolds (diameter 20 mm, n = 5) in 12-
well tissue culture plates were sterilised by immersion
in 70 % ethanol and exposure to UV irradiation for 1 h,
washed three times with diH2O, and seeded with 1×104
cells. The scaffolds were stabilised in the bottom of the
wells by placing sterilised stainless steel rings (20 mm
diameter, a generous gift from Dr. Il Keun Kwon, Kyung
Hee University, Seoul, Korea) over the scaffolds’ outer
edge. The cell-laden nanobres were cultured in 2 mL
media as described above.
To investigate the morphological changes of the
cultured cells on the scaffolds, samples were xed in
3.7 % formaldehyde in phosphate buffered saline (PBS) for
10 min and then permeabilised in cold cytoskeleton buffer
Fig. 3. Scanning electron micrographs of electrospun unmodied
methacrylated alginate (UMA), RGD-modied methacrylated
alginate (RMA), as well as heparin and RGD-modied methacrylated
alginate (HRMA) nanobres (prior to PEO extraction) (a-f) before
and (g-l) after cross-linking using UV irradiation. Images represent:
(a and g) UMA14 (b and h) UMA24 (c and i) UMA44, (d and
j) UMA84, (e and k) RMA84, and (f and l) HRA84. Scale bars
represent 3 μm.

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SI Jeong et al. Biodegradable alginate nanobre scaffolds
(50 mM NaCl, 150 mM sucrose, 3 mM MgCl2, 50 mM Tris
base, 0.5 % Triton X-100) for 5 min at 4 °C. The samples
were incubated in blocking buffer (1 % bovine serum
albumin (BSA), 0.1 % Tween-20, 0.02 % sodium azide in
PBS) for 60 min at 37 °C. Following a wash in PBS, the
samples were incubated for 60 min at 37 °C in blocking
buffer containing rhodamine-phalloidin at a 1:200 dilution
(Invitrogen/Life Technologies, Carlsbad, CA, USA) to
stain for F-actin microlaments, and Hoechst 33258 at
a 1:1000 dilution (Invitrogen/Life Technologies) to stain
nuclear DNA. Following a gentle wash in PBS, samples
were mounted in Fluoromount Aqueous Mounting Medium
(Sigma-Aldrich) on glass slides. The samples were then
visualised on a Nikon inverted uorescence microscope
(ECLIPSE TE 300, Nikon, Tokyo, Japan). Digital images
were acquired using a digital camera (Retiga-SRV,
QImaging, Burnaby, BC, Canada).
For quantication of changes in cell number after 1,
3 and 7 days of culturing, the samples were transferred
to a new 12-well plate, and 1 mL of a 20 % CellTiter
96 Aqueous One Solution (Promega, Madison, WI,
USA) which contains 3-[4,5-dimethylthiazol-2-yl]-5-
[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-
tetrazolium (MTS-tetrazolium) was added. The MTS-
tetrazolium compound can be metabolised by mitochondria
in living cells into a coloured formazan product that is
soluble in cell culture medium. After incubating at 37 °C
for 90 min, 100 μL of each solution was transferred to a
96-well plate and the absorbance at 490 nm was determined
using a plate reader (SAFIRE, Tecan, Durham, NC, USA).
Growth factor release from the nanobrous scaffolds
The release of FGF-2 from the photo-crosslinked
alginate nanofibrous scaffolds was determined. The
photo-crosslinked, PEO-extracted, lyophilised scaffolds
(diameter 20 mm, n = 5) were incubated with 100 ng
FGF-2 (for HRMA84 scaffolds) or 100 ng FGF-2 mixed
with 2.5 μg heparin (for UMA84 and RMA84 scaffolds)
in PBS for 1 h at 4 °C. When the growth factor solution
was incubated with the nanobrous scaffolds, the solution
was completely absorbed into the scaffolds within 1 h. The
samples were then transferred to Transwell membranes
(Corning, Corning, NY, USA) in 6-well tissue culture plates
and incubated at 37 °C in 5 mL DMEM for 14 days, with
the media replaced at days 1, 2, 3, 5, 7, 10 and 14. The
amount of FGF-2 released into the media was determined
Fig. 4. Scanning electron micrographs of photo-cross-linked UMA84, RMA84, and HRMA84 nanobres following
PEO extraction in (a-c) diH2O and (d-f) DMEM at 37 °C for 5 days. Images represent: (a and d) UMA84, (b and
e) RMA84, and (c and f) HRMAP84. Scale bars represent 6 μm.
Fibre diameters (nm) Before cross-linking After cross-linking After PEO extraction
UMA84 195.4 ± 23 183.2 ± 27 256.3 ± 43
RMA84 185.4 ± 27 190.4 ± 30 297.9 ± 42
HRMA84 185.5 ± 37 182.2 ± 36 278.2 ± 40
Table 2. The bre diameters of electrospun UMA, RMA, and HRMA nanobres (prior to PEO extraction)
before and after UV irradiation, and photo-cross-linked UMA, RMA, and HRMA nanobres following PEO
extraction. Values are average ± SD.

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SI Jeong et al. Biodegradable alginate nanobre scaffolds
with an ELISA assay as per manufacturer’s instructions
(Duoset, R&D Systems, Minneapolis, MN, USA).
To evaluate the activity of the encapsulated FGF-2
on cells, photo-crosslinked, PEO-extracted, lyophilised
scaffolds (n = 5) were incubated with 100 ng FGF-2 (for
HRMA84 scaffolds) or 100 ng FGF-2 mixed with 2.5 μg
heparin (for UMA84 and RMA84 scaffolds) in PBS for
1 h at 4 °C. These scaffolds were again placed in Transwell
membranes in 6-well plates, with 1x104 HDFs seeded in
the wells and cultured in 5 mL media as described above.
A control population consisting of cells seeded in wells
but cultured without scaffolds was examined. The change
in cell number over time was measured indirectly using
an MTS assay as previously described.
Statistical analysis
Data are expressed as mean ± SD. Statistical analysis was
carried out using ANOVA (InStat 3, GraphPad Software,
La Jolla, CA, USA), and a value of p < 0.05 was considered
statistically signicant.
Results
UMA, RMA, and HRMA were blended with PEO
and electrospun to form nanofibrous scaffolds. The
polymer solutions examined for electrospinning MA/
PEO nanobres are listed in Table 1. To demonstrate that
the photo-crosslinkable alginate could be electrospun,
SEM photomicrographs of photo-crosslinked MA/
PEO nanobres before and after crosslinking with UV
irradiation for 10 min were obtained (Fig. 3). Before UV
irradiation, some of the conditions (MA wt% of 1 or 2)
resulted in nanobres with beaded structures (Fig. 3a,b).
Increasing the concentration of alginate to 4 wt% resulted
in nanobres with minimal beaded structures (Fig. 3c).
Uniform nanobres were obtained using UMA, RMA, and
HRMA at 8.0 wt% of MA and 4.0 wt% PEO (Fig. 3d-f).
The electrospun crosslinked UMA, RMA, and HRMA
scaffolds were soaked in diH2O or DMEM for 5 days to
leach out the water-soluble and uncrosslinked PEO. After
PEO extraction in diH2O, the photo-crosslinked MA
scaffolds lost their nanobrous structure due to swelling
(Fig. 4a-c). However, when the nanobres were soaked
in DMEM to extract the PEO, the scaffolds maintained a
nanobrous structure (Fig. 4d-f).
The average bre diameters of MA scaffolds before
and after photo-crosslinking were measured using the SEM
images to determine if the crosslinking or heparin or RGD
modication affected the bre nanostructure (Table 2).
There was a slight decrease in the bre diameters of the
scaffolds made with unmodied alginate after crosslinking,
and no signicant difference before and after crosslinking
for the RMA or HRMA scaffolds. Others have also reported
Fig. 5. (a) Representative stress-strain curves, (b) elongation at break, (c) tensile strength, and (d) Young’s moduli
of photo-cross-linked UMA84, RMA84, and HRMA84 nanobres (prior to PEO extraction). *p < 0.05

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SI Jeong et al. Biodegradable alginate nanobre scaffolds
no change in the bre diameters of photo-crosslinked
nanobres using UV irradiation (Jin et al., 2008). The bre
diameters increased upon extraction of the PEO in DMEM,
due to some swelling of the nanobres in the solution.
However, these results indicate that the scaffolds maintain
a nanobrous structure even upon soaking in DMEM for
5 days.
Constant strain rate tensile tests were performed to
determine the mechanical properties of the electrospun
photo-crosslinked alginate nanofibre scaffolds before
PEO extraction, specifically elongation at break,
tensile strength and Young’s modulus (Fig. 5). The
addition of methacrylated heparin to the scaffolds was
found to inuence their mechanical properties as can
be seen from representative stress-strain curves (Fig.
5a). The elongation at break for UMA and RMA was
4.04 ±0.90 % and 3.67 ±0.75 %, respectively, and for
HRMA it decreased even further to 2.46 ±0.62 % (Fig.
5b); HRMA had a signicantly lower elongation at break
than UMA. The tensile strength and Young’s modulus of
HRMA (2.22 ±0.39 and 1.04 ±0.11 MPa, respectively)
were signicantly greater than UMA (1.35 ±0.14 and
0.44 ±0.02 MPa) and RMA scaffolds (1.42 ±0.12 and
0.53 ±0.06 MPa), indicating the heparin reinforced the
nanobres (Fig. 5c,d).
ATR-FTIR was used to examine the chemical
composition of the nanobres and verify the removal
of PEO following 5 days extraction in media. The
characteristic peaks for pure PEO were observed at 844,
963, 1103 and 1343 cm-1 (Fig. 6a) (Ji et al., 2006). The
carboxylic acid group of alginate was detected at 1616
cm-1 in pure alginate (Fig. 6b) and the photo-crosslinked
alginate nanobres before and after PEO extraction (Fig.
6c-f)(Lu et al., 2006). Note that the peaks characteristic of
PEO are absent in the spectra for scaffolds that have been
leached of PEO (Fig. 6d-f).
The stability and degradation of the nanobres over
time was studied by incubating the photo-crosslinked,
PEO-extracted nanobrous scaffolds in DMEM at 37 °C
for an additional 3 weeks beyond the 5 days required for
PEO leaching, and then examining their morphologies
with SEM. The scaffolds maintained their nanobrous
structure after 1 week of incubation (Fig. 7a-c), but lost
this structure after 2 and 3 weeks of incubation (Fig.
7d-i). To examine cellular interactions with the photo-
crosslinked, PEO-extracted scaffolds, HDFs were seeded
and cultured on them for 7 days (Fig. 8). F-actin and nuclei
staining of the cells at 1 and 7 days revealed that the few
HDFs on UMA nanobres remained rounded and did not
proliferate substantially, while those on the RMA and
HRMA nanobres spread and proliferated (Figure 8a-f).
The mitochondrial activity of the HDFs cultured on the
nanobre scaffolds was used as an indirect measure of
changes in cell number over time, as assessed by an MTS
assay (Fig. 8g).
It was hypothesised that the heparin modication of
the photo-crosslinkable alginate would provide sustained
release of heparin-binding growth factors from the
electrospun nanobres. Therefore, the release proles
of FGF-2 from photo-crosslinked, PEO-extracted UMA,
RMA, and HRMA nanobre scaffolds was examined
(Fig. 9a). Indeed, it was shown that the release of this
growth factor was more sustained from the nanobres
containing the heparin-modied alginate. FGF-2 has a
mitogenic effect on cells (Chen et al., 2004), and thus
the bioactivity of the released FGF-2 was examined by
observing its effect on cell proliferation indirectly through
MTS assay (Fig. 9b). Cells cultured with the UMA, RMA,
and HRMA scaffolds for 1 and 4 days show similar levels
of proliferation. At 7 days, there were signicantly more
cells cultured with the UMA and RMA scaffolds compared
to those in media control without FGF-2, indicating that the
bioactivity of the FGF-2 is preserved in these scaffolds. For
the cells cultured with the HRMA scaffolds, by 4 days the
cells showed signicantly greater proliferation than those
cultured in media, and by 7 days the cells cultured with the
HRMA scaffolds exhibit a signicantly higher degree of
proliferation than those cultured with the UMA scaffolds
or in media.
Discussion
Electrospinning can be used to form nanofibrous
biopolymer scaffolds; these types of scaffolds have unique
features including a high surface area to volume ratio and
substantial interconnected pores. While alginate in its
native form has been electrospun (Bhattarai et al., 2006;
Lu et al., 2006; Jeong et al., 2010), electrospinning photo-
polymerisable alginate into stable nanobres has not been
demonstrated. Due to the benets that photo-crosslinkable
Fig. 6. ATR-FTIR
spectra of electrospun
nanobres composed
of photo-cross-linked
alginate blended with
PEO. (a) PEO alone,
(b) pure alginate, (c)
HRMA84 nanobres,
(d) PEO-extracted
UMA84, (e) PEO-
extracted RMA84,
(f) PEO-extracted
HRMA84. White
arrows indicate a
characteristic peak of
alginate; black arrows
indicate PEO peaks.

338 www.ecmjournal.org
SI Jeong et al. Biodegradable alginate nanobre scaffolds
alginate can provide, including tailorable degradation,
mechanical and cell adhesive properties and the ability
to covalently incorporate methacrylated heparin into
the crosslinked matrices for controlled protein delivery,
it was important to ascertain whether this biomaterial
could be electrospun into nanobres and then photo-
crosslinked to maintain its nanobrous structure. The
goal of this work was threefold: (1) to demonstrate the
capacity of methacrylated alginate to be electrospun into
a nanobrous matrix and subsequently photo-crosslinked,
(2) to determine if cell adhesion and proliferation could
be enhanced on these matrices by covalent modication
with cell adhesion peptides, and (3) to incorporate heparin
into the hydrogels during the photopolymerisation process
to provide sustained release of bioactive heparin binding
growth factors and examine their potential for accelerating
cell proliferation on the matrices.
Alginate cannot be spun by itself, most likely because
of a lack of chain entanglements (Nie et al., 2008).
Therefore, it is typically electrospun in the presence of PEO
or another biocompatible polymer, which when blended
with alginate promotes the formation of uniform nanobres
during electrospinning (Jeong et al., 2010). Here, we have
demonstrated that a blended solution of methacrylated
alginate (which can be crosslinked by exposure to UV light)
and PEO could be electrospun to form uniform nanobres.
The UV crosslinking and presence of RGD peptide or
heparin had no visible effect on the morphologies of the
nanobres.
The mechanical properties of these nanobres were
measured by tensile testing. Interestingly, the presence of
heparin in these scaffolds serves to change the mechanical
properties, as evidenced by a statistically signicant lower
elongation at break (compared to the UMA scaffolds),
higher tensile strength and higher Young’s modulus
(compared to both the UMA and RMA scaffolds). It
is likely that the methacrylated heparin and PEO are
interacting, possibly by hydrogen bonding between PEO
and the carboxyl group of the heparin, in the nanobres to
provide these modied mechanical properties, as has been
previously suggested for crosslinked networks of heparin
and pluronic (Lee et al., 2010).
Although PEO was necessary for the electrospinning
of the alginate, it is possible to have nanofibrous
scaffolds comprised of alginate and/or alginate-heparin
alone by leaching out the PEO; removing the inert PEO
would provide cells a greater degree of contact with the
biologically active modied polysaccharide. Therefore,
using FTIR, it was demonstrated that the PEO could be
successfully removed from these nanobres to leave ALG
Fig. 7. The degradation of photo-cross-linked PEO-extracted UMA84, RMA84, and HRMA84 nanobres incubated
in DMEM at 37 °C for (a-c) 1 week, (d-f) 2 weeks, and (g-i) 3 weeks. Scale bars represent 3 μm.

339 www.ecmjournal.org
SI Jeong et al. Biodegradable alginate nanobre scaffolds
or HP-ALG nanobres. In our previous report, photo-
crosslinked ALG and HP-ALG bulk hydrogels were stable
in media for 8 weeks, but lost approximately 20 % of their
mass by 2 weeks (Jeon et al., 2011). However, the photo-
crosslinked alginate nanobre scaffolds presented here
degraded faster than bulk hydrogels. It is likely that the
scaffolds degraded more rapidly due to the high surface-
to-volume ratio of the nanobres, which would allow
hydrolysis to occur on more of the scaffolds at earlier
time-points compared to bulk hydrogels. Regardless, it
is during this initial 1-2 weeks that increased surface-to-
volume ratio of the material and therefore increased cell
biomaterial interactions would likely have their maximal
impact. For example, the presentation of adhesion ligands
on the scaffold, such as the RGD-containing ligand in this
study, to control cellular behaviour will provide the greatest
inuence at early time points prior to the cells secreting
their own ECM. The scaffolds themselves are stable for at
least 3 weeks, beyond the time point at which the nanoscale
structure is lost.
The interconnected pores of a nanobrous scaffold are
important for cell inltration and proliferation. Although
the scaffolds soaked in diH2O swelled substantially and
lost much of their porous nature, the photo-crosslinked
Fig. 8. (a-f) Fluorescence photomicrographs of HDFs cultured on photo-cross-linked PEO-extracted UMA84, RMA84,
and HRMA84 nanobre scaffolds stained with rhodamine-phalloidin and DAPI. Scale bars represent 100 μm. (g)
MTS assay of HDFs cultured on these nanobre scaffolds for 1, 3 and 7 days. *p < 0.05.

340 www.ecmjournal.org
SI Jeong et al. Biodegradable alginate nanobre scaffolds
Fig. 9. (a) Cumulative release
proles of FGF-2 from photo-
cross-linked, PEO-extracted
UMA84, RMA84, and HRMA84
nanobre scaffolds for 14 days
and (b) MTS assay demonstrating
the bioactivity of FGF-2 released
from the photo-cross-linked,
PEO-extracted UMA84, RMA84,
and HRMA84 scaffolds.
*p < 0.05.
nanobres soaked in DMEM have a porous structure
(Fig. 4d-f) that may promote cell interactions with the
scaffolds. The increased swelling of photo-crosslinked MA
in diH2O compared to DMEM is likely osmosis driven,
and has been reported in our previous work (Jeon et al.,
2010). Although alginate is non-adhesive to cells, it can be
chemically modied with cell adhesive peptide sequences
to promote cell adhesion, migration, and proliferation in the
scaffolds (Rowley et al., 1999). There were signicantly
more HDFs on the nanofibres modified with the cell
adhesive peptide containing the RGD sequence compared
to the unmodied alginate at all time-points, and the HDFs
exhibited increased proliferation on the RGD-modied
nanobres. These ndings demonstrate that the GRGDSP
modication of the photo-crosslinkable alginate promotes
cell adhesion, and in turn, proliferation.
Native ECM protects and sequesters growth factors
(Benoit and Anseth, 2005; Schultz and Wysocki, 2009).
The afnity of heparin and FGF-2 has been reported
based on the electrostatic binding between the negatively
charged sulphonyl and carboxyl groups of heparin and
positively charged amino groups of FGF-2 (Raman et al.,
2003; Schultz and Wysocki, 2009). Other groups have
demonstrated release of FGF-2 from different heparin-
functionalised scaffolds (Benoit and Anseth, 2005; Guan
et al., 2007; Sakiyama-Elbert and Hubbell, 2000; Shen
et al., 2011; Wu et al., 2011). In this report, the release
kinetics of FGF-2 from electrospun photo-crosslinked
alginate bres was quantied to determine if heparin
could modulate FGF release and activity in this system.
While the FGF-2 showed a burst release from the UMA
and RMA nanobres on the rst day followed by little
subsequent release, its release from the HRMA scaffolds
was sustained over the course of one week. This indicates
that the heparin in these scaffolds provides afnity binding
of the growth factor, allowing it to remain within the
scaffolds for a longer period of time before being released
to the surrounding environment. Furthermore, the released
FGF-2 was bioactive and capable of inuencing cellular
proliferation. Due to its sustained presentation to these
cells, the growth factor released from the HRMA scaffolds
enhanced the proliferation of the HDFs over time compared
to that released from UMA or RMA scaffolds. These data
demonstrate the importance that controlled and prolonged
presentation of bioactive factors can have on regulating
cell behaviour for tissue regeneration applications.

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SI Jeong et al. Biodegradable alginate nanobre scaffolds
Conclusion
In this study, photo-crosslinked alginate nanobre scaffolds
were prepared by electrospinning. The scaffolds were
comprised of uniform nanobres which crosslinked to
form a porous structure. The photo-crosslinkable alginate
could be modied with cell adhesive peptide sequences
and/or with heparin to confer additional bioactivity to the
scaffolds. The covalent coupling of a peptide containing the
RGD cell adhesive sequence, which is found in bronectin
and other ECM molecules (Ruoslahti and Pierschbacher,
1987; Sechler et al., 1997), promoted the adhesion of HDFs
to these nanobrous scaffolds and subsequent proliferation
over time; these are promising initial data for the use of
this system in skin tissue engineering. It may be possible
to tailor these scaffolds for use with other cell types for
other applications through the use of other bioactive
peptide sequences, for example sequences such as YIGSR
or IKVAV, which are found in laminin (Graf et al., 1987;
Tashiro et al., 1989). Additionally, it was demonstrated
that by covalently modifying the alginate with heparin,
FGF-2 was released in a sustained manner over the course
of one week from these scaffolds, and the released growth
factor retained its bioactivity as demonstrated by enhanced
proliferation of HDFs. These scaffolds could also be used
to achieve sustained release of different heparin-binding
growth factors for other regenerative medicine pursuits.
The release of heparin-binding growth factors might be
further regulated by altering the concentration of heparin
used in the scaffolds. In future studies, the degradation
rate and mechanical properties of these nanobres could
be controlled by, for example, varying the degree of
methacrylation, the molecular weight of the alginate, or
by oxidising the alginate. In summary, these electrospun,
biodegradable nanobres composed of photo-crosslinkable
alginate are promising for use as scaffolds in wound
healing and tissue regeneration, as their physical (i.e.,
nanostructure, modulus and degradation rate) and cell
adhesive properties and growth factor release proles may
be tailored for specic applications.
Acknowledgements
The authors gratefully acknowledge funding from the
National Institutes of Health (AR063194, DE022376,
AR061265), the AO Foundation, a Biomedical Research
and Technology Transfer Grant 08-081 from the Ohio
Department of Development, and a New Scholar in Aging
grant from the Ellison Medical Foundation.
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Discussion with Reviewer
Reviewer II: The material is proposed as a scaffold for
wound healing or regeneration of skin, but in fact all that
has been done here is to grow some dermal broblasts
on the surface. There is no indication that they produce
any kind of extracellular matrix or go any further towards
generating a new tissue than they would if they were grown
in a plate. How do the authors propose to use this material
to regenerate tissue, as opposed to just growing cells?
Authors: In order to regenerate wounded skin tissue,
a dermal substitute must provide an environment that
allows dermal broblasts to adhere, proliferate and deposit
extracellular matrix just as they would in native tissue. As
mentioned earlier, cells are not able to adhere to alginate
naturally. With respect to investigating the inuence of
these nanobres on regulating cellular behaviour, the
rst goal was to determine whether photo-polymerisable
alginate could be electrospun into uniform nanobres and

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SI Jeong et al. Biodegradable alginate nanobre scaffolds
then whether controlled RGD adhesion ligand presentation
would enhance cell-biomaterial interactions with the
nanobre scaffolds. Following successful formation of
stable nanobres, human dermal broblasts were seeded
on the surface of the scaffolds and cultured for seven days.
Signicantly greater cell adhesion and proliferation was
quantied on the RGD-modied materials compared to
the unmodied. The second goal of this manuscript was
to determine if controlled delivery of FGF-2 from the
scaffolds with or without adhesion peptide modication
could enhance human dermal broblast proliferation.
By day seven, all scaffolds releasing FGF-2 resulted in
increased cell number compared to the control, and there
was increased cell number in the heparin-modied scaffold
condition compared to the unmodied scaffold condition.
The ability to engineer these nanobrous scaffolds to
control cell adhesion and growth factor presentation, and
thus inuence cellular behaviours such as proliferation,
demonstrates that indeed we are able to create a unique
scaffold that can be tuned to regulate cellular function
for the regeneration of specic tissues such as skin. In
summary, we were able to successfully electrospin photo-
crosslinkable alginate into uniform nanobres that offer
controllable physical and biochemical properties and
growth factor release capacity and thus may have great
utility in wound healing. We recognise that this is a starting
point for full development of a biomaterial technology
that is capable of driving skin tissue regeneration, but
this manuscript presents critical data demonstrating the
potential of this new system for incorporation of specic
physical and biochemical signals in a modular manner for
regulating cell behaviour.