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SDF-1α gene-activated collagen scaffold enhances provasculogenic response in a coculture of human endothelial cells with human adipose-derived stromal cells

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Novel biomaterials can be used to provide a better environment for cross talk between vessel forming endothelial cells and wound healing instructor stem cells for tissue regeneration. This study seeks to investigate if a collagen scaffold containing a proangiogenic gene encoding for the chemokine stromal-derived factor-1 alpha (SDF-1α GAS) could be used to enhance functional responses in a coculture of human umbilical vein endothelial cells (HUVECs) and human adipose-derived stem/stromal cells (ADSCs). Functional responses were determined by (1) monitoring the amount of junctional adhesion molecule VE-cadherin released during 14 days culture, (2) expression of provasculogenic genes on the 14th day, and (3) the bioactivity of secreted factors on neurogenic human Schwann cells. When we compared our SDF-1α GAS with a gene-free scaffold, the results showed positive proangiogenic determination characterized by a transient yet controlled release of the VE-cadherin. On the 14th day, the coculture on the SDF-1α GAS showed enhanced maturation than its gene-free equivalent through the elevation of provasculogenic genes (SDF-1α—7.4-fold, CXCR4—1.5-fold, eNOS—1.5-fold). Furthermore, we also found that the coculture on SDF-1α GAS secretes bioactive factors that significantly (p < 0.01) enhanced human Schwann cells’ clustering to develop toward Bünger band-like structures. Conclusively, this study reports that SDF-1α GAS could be used to produce a bioactive vascularized construct through the enhancement of the cooperative effects between endothelial cells and ADSCs.
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Journal of Materials Science: Materials in Medicine (2021) 32:26
https://doi.org/10.1007/s10856-021-06499-6
TISSUE ENGINEERING CONSTRUCTS AND CELL SUBSTRATES
Original Research
SDF-1αgene-activated collagen scaffold enhances provasculogenic
response in a coculture of human endothelial cells with human
adipose-derived stromal cells
Ashang L. Laiva 1,2 Fergal J. OBrien1,3,4 Michael B. Keogh1,2
Received: 15 June 2020 / Accepted: 17 February 2021 / Published online: 6 March 2021
© The Author(s) 2021
Abstract
Novel biomaterials can be used to provide a better environment for cross talk between vessel forming endothelial cells and
wound healing instructor stem cells for tissue regeneration. This study seeks to investigate if a collagen scaffold containing a
proangiogenic gene encoding for the chemokine stromal-derived factor-1 alpha (SDF-1αGAS) could be used to enhance
functional responses in a coculture of human umbilical vein endothelial cells (HUVECs) and human adipose-derived stem/
stromal cells (ADSCs). Functional responses were determined by (1) monitoring the amount of junctional adhesion molecule
VE-cadherin released during 14 days culture, (2) expression of provasculogenic genes on the 14th day, and (3) the
bioactivity of secreted factors on neurogenic human Schwann cells. When we compared our SDF-1αGAS with a gene-free
scaffold, the results showed positive proangiogenic determination characterized by a transient yet controlled release of the
VE-cadherin. On the 14th day, the coculture on the SDF-1αGAS showed enhanced maturation than its gene-free equivalent
through the elevation of provasculogenic genes (SDF-1α7.4-fold, CXCR41.5-fold, eNOS1.5-fold). Furthermore, we
also found that the coculture on SDF-1αGAS secretes bioactive factors that signicantly (p< 0.01) enhanced human
Schwann cellsclustering to develop toward Bünger band-like structures. Conclusively, this study reports that SDF-1αGAS
could be used to produce a bioactive vascularized construct through the enhancement of the cooperative effects between
endothelial cells and ADSCs.
Graphical Abstract
*Michael B. Keogh
mkeogh@rcsi-mub.com
1Tissue Engineering Research Group, Department of Anatomy and
Regenerative Medicine, Royal College of Surgeons in Ireland, 123
St. Stephens Green, Dublin 2, Ireland
2Department of Biomedical Science, Royal College of Surgeons in
Ireland, Adliya, Bahrain
3Trinity Centre for Bioengineering, Trinity Biomedical Sciences
Institute, Trinity College Dublin, Dublin 2, Ireland
4Advanced Materials and Bioengineering Research Centre, Royal
College of Surgeons in Ireland and Trinity College Dublin,
Dublin, Ireland
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1 Introduction
Insufcient regeneration of a vascular network remains one
of the major limitations of biomaterials scaffolds used in
wound healing [1]. To overcome this problem, functiona-
lizing the scaffolds with either natural or synthetic proan-
giogenic growth factors to promote local angiogenesis is a
popular strategy [25]. These scaffolds are designed to
generate a gradient of growth factors to stimulate vessel
growth toward the desired site and promote their maturation
[6]. However, there is a lack of general consensus on the
optimal dose of growth factors to be delivered. In addition,
these angiogenic factors are susceptible to proteolysis, while
bolus delivery of high doses to sustain the therapeutic effect
raises the risk of toxicity as well as cost [7]. Parallel
investigations such as prevascularizing biomaterial scaf-
folds using endothelial cells show potential [810]. Appli-
cation of these prevascularized scaffolds is expected to
signicantly promote biomaterial integration as well as
accelerate vascular regeneration by anastomising with host
vessels [11,12]. However, maintaining the stability of the
vascular structure within the scaffold remains an important
developmental criteria for therapeutic success. Conse-
quently, strategies aiming at improving the stability of the
endothelial network while allowing adequate angiogenesis
continues to be investigated [13,14].
One of the strategies adopted in our group for enhancing
the stability of the endothelial network is the delayed
addition of stem cells to a preformed endothelial network.
This approach has also been found to enhance vascular-
ization in vivo [10]. Having observed the importance of
cross talk between endothelial cells and stem cells, we
hypothesized that the endothelial cells could be activated to
recruit surrounding stem cells and enhance their interac-
tion. Biomaterial scaffolds functionalised with therapeutic
genes, called the gene-activated scaffolds (GAS), are an
effective platform for activating cells. It works by inducing
overexpression of the therapeutic protein by the cells,
eventually aiding in enhancing the regenerative capacity of
the scaffold [1517].
Our group focuses in the development of nonviral based
GAS through the use of nonviral vectors such as poly-
ethyleneimine (PEI) [16,18,19]. Using this vector-based
GAS, we have shown that the delivery of a proangiogenic
gene stromal-derived factor-1 alpha (SDF-1α) to mesench-
ymal stem cells (MSCs) signicantly potentiates its angio-
genic action on endothelial cells [20]. Moreover, SDF-1αis
a potent chemokine upregulated during the early stages of
wound healing. It is crucial for the homing of endothelial
progenitor cells to ischemic sites and promote local angio-
genesis [2124]. Therefore, in this study, we seek to
investigate if the SDF-1αGAS could be used to enhance the
provasculogenic maturation of cocultures of endothelial cells
and adipose-derived stem/stromal cells (ADSCs). The pre-
vious study [10] from our group used human bone marrow
MSCs to reinforce the endothelial network. However, in our
study, we seek to use human stem/stromal cells derived from
the adipose tissue (ADSCs) because of the relative abun-
dance of bone marrow MSCs, ease of isolation, and good
patient compliance [25]. Several studies have also shown
that ADSCs possess strong vasculogenic properties as well
as demonstrate great potential for enhancing vascularization
of various scaffolds by endothelial cells [2529].
Nevertheless, in addition to vasculogenesis, neuronal
regeneration is an important aspect of wound healing that is
often overlooked. Schwann cells (SCs) are the key media-
tors of neurogenesis [30]. During angiogenesis, the angio-
genic vessels guide the SCs to the site of wound [31]. The
SCs then lay down matrix to guide regenerating axons and
promote their reinnervation of the epidermis [32]. More-
over, SCs also possess the ability to provide contact gui-
dance to endothelial cells and direct their morphogenesis
toward a proangiogenic growth [33].
Therefore, the specic aims of this study are to deter-
mine if (1) the transfection of SDF-1αgene induces the
secretion of SDF-1αproteins by human endothelial cells
and that the protein possess chemotactic activity on human
ADSCs; (2) SDF-1αGAS can support angiogenesis and
promote provasculogenic response in endothelial cells and
its coculture with ADSCs; and ultimately if (3) the vas-
cularized construct can signal human SCs toward a pro-
neurogenic response.
2 Materials and methods
2.1 Plasmid propagation and preparation of
polyplex
Plasmid DNA (pDNA) encoding for the therapeutic gene
SDF-1α(pSDF-1α) was obtained from InvivoGen, San
Diego, USA. The plasmids were rst amplied by trans-
forming chemically competent DH5αE. coli cells (Bios-
ciences, Ireland) according to the manufacturers protocol.
Transformed cells were then expanded in lysogeny broth
(LB) plates containing 100 μg/ml of blasticidin as the
selective antibiotic for pSDF-1α. After 24 h at 37 °C, bac-
terial colonies were harvested and amplied in LB broth
containing the appropriate antibiotic and cultured overnight
in a shaker incubator at 37 °C. Plasmid purication was
performed using a QIAGEN®EndoFree®Plasmid Maxi kit
(Qiagen, Sussex, UK) and nal nucleic acid concentration
was determined using NanoDrop 1000 spectroscopy. Plas-
mids were further diluted in TE buffer to obtain a working
concentration of 0.5 μg/μl and stored at 20 °C until use.
pDNA encoding a nontherapeutic Gaussia luciferase (pLuc)
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purchased from New England Biolabs, Massachusetts,
USA, was similarly amplied using ampicillin as the
selective antibiotic. Based on our previous study, polyplex
particles were formulated by initially mixing a specied
amount of branched cationic 25 kDa PEI (Sigma-Aldrich,
Ireland) and anionic pDNA (xed at a dose of 2 μg) to give
an N/P ratio of 10.
2.2 Expansion and transfection of cells
Human umbilical vein endothelial cells (HUVECs) and
ADSCs were obtained from Cell Applications, Inc. and
iXCells Biotechnologies, USA, respectively. HUVECs
were cultured in supplemented EndoGRO media (EGM-2;
Merck Millipore, USA) without VEGF (EGMVEGF) and
expanded to passage 4 for all experiments. ADSCs were
expanded in 1:1 Dulbeccos Modied Eagles Medium/
Nutrient mixture F-12 (D8437, Sigma-Aldrich, UK) sup-
plemented with 10% FBS (Gibco, UK), 2% penicillin/
streptomycin (Sigma-Aldrich, UK), and 1% amphotericin B
(Gibco, UK) and harvested at passage 5 for subsequent
experiments. The HUVECs were seeded at a density of 5 ×
104cells per well in six-well adherent plates (Corning,
Costar, UK) 24 h prior to transfection. One hour prior to
transfection, the media were removed and replaced with
Opti-MEM (Gibco, UK). Meanwhile, PEI was mixed with a
2 µg dose of pDNA at an N/P ratio of 10 and was allowed to
assemble into polyplex by electrostatic interaction for
30 min. The PEIpDNA polyplexes were then suspended in
Opti-MEM and added to the monolayer and incubated at
37 °C for 15 min. After 15 min, an additional 1 ml of Opti-
MEM was added and the cells were incubated for ~4 h.
After this transfection period, the medium was removed and
the cell monolayer was rinsed with PBS. EGMVEGF was
then added and the cells were incubated at 37 °C to allow
expression of the transgene. Media change was performed
by collecting 1 ml of the media (conditioned media (CM))
and replacing it with fresh media at 3, 7, 10, and 14 days.
All CM were stored at 80 °C until analysis.
2.3 Effect of transfection on viability of cells
To determine the effect of therapeutic gene transfer on the
transfected cell, cell viability following transfection with
PEIpSDF-1αor PEIpLuc was assessed using the colori-
metric MTS assay (CellTiter 96®AQueous One Solution,
Promega, Madison, WI, USA). Briey at 1, 3, and 7 days
post transfection, 20 μl of the MTS reagent was added to the
cells in 100 μl of media, and incubated for 4 h at 37 °C.
Intensity of the resulting color was measured at an absor-
bance of 490 nm using a Multiskan GO plate reader
(Thermo Scientic, UK). Cell viability percentage (%) was
determined according to the equation (absorbance[transfected]/
absorbance[control]) × 100, keeping the untransfected cells as
100% viability control.
2.4 ELISA for quantication of SDF-1αprotein
production post transfection in 2D culture
In order to determine successful transfection of pSDF-1α,
secretion of SDF-1αproteins into the culture medium was
determined using a human SDF-1αspecic ELISA kit
(DY350, R&D Systems, UK) according to the manu-
facturers instructions. One hundred microliters of CM
collected from days 3, 7, 10, and 14 were used for the assay
and the absorbance was read at 450 nm using a Multiskan
GO plate reader (Thermo Scientic, UK) whereby the
quantity of SDF-1αprotein present was deduced by calcu-
lating against a standard curve.
2.5 Coculture of HUVECs and ADSCs in a 2D setup
In order to assess the functionality of secreted proteins, a
delayed addition approach previously reported by our group
[10] was adopted where stem cells are added onto preseeded
HUVECs. Based on the previous study [10], the ratio of
HUVECs to the ADSCs was set at 4:1. The transfection
experiment was performed as in the section Expansion and
transfection of cells,except that HUVECs were initially
seeded at a density of 4 × 104and allowed to grow until the
3rd day, after which 1 × 104ADSCs were added. On the
14th day of incubation, images of the cocultures were
captured using a Celestron®digital microscope imager,
attached to a phase-contrast Olympus CKX31 microscope.
CM was also collected from these groups as described in the
section Expansion and transfection of cells,to analyze
how the expression of SDF-1αis affected by the addition of
ADSCs. All the cocultures were maintained in EGMVEGF.
2.6 Cell seeding on SDF-1αGAS
SDF-1αGAS was developed as described earlier [20],
which basically involves soak-loading of the PEIpSDF-1α
polyplex nanoparticles into a freeze-dried porous 3D coll-
CS scaffold [34]. For this study, a double-sided cell seeding
of equal cellular density was performed. For culture of
HUVECs alone, a total of 5 × 105cells were seeded onto the
scaffold/SDF-1αGAS, while for coculture groups, 4 × 105
HUVECs were initially seeded and allowed to endothelia-
lize the scaffolds/SDF-1αGAS for 3 days before 1 × 105
ADSCs were added onto it [10]. Consequently, four groups
were developed (1) HUVECs alone on gene-free scaffold,
(2) HUVECs alone on SDF-1αGAS, (3) coculture on gene-
free scaffold, and (4) coculture on SDF-1αGAS. For initial
transfection of HUVECs in the SDF-1αGAS, 2 ml of
Opti-MEM was added and the plates were incubated at
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Content courtesy of Springer Nature, terms of use apply. Rights reserved.
37 °C for 24 h after which the media were removed and
replaced with 2 ml of EGMVEGF. To assay the temporal
regulation of soluble vascular molecules, media were col-
lected and replaced in the same manner as described pre-
viously in the section Expansion and transfection of cells.
2.6.1 ELISA for quantication of soluble vascular
endothelial cadherin (VE-cadherin) and VCAM-1 in
endothelialized SDF-1αGAS
To determine angiogenic growth of the endothelial cultures
in SDF-1αGAS, secretion of the soluble forms of endo-
thelial junctional adhesion molecule VE-cadherin (DY938,
R&D Systems, UK) and inammation induced transmem-
brane molecule VCAM-1 (DY809, R&D Systems, UK) was
analyzed. To assay angiogenesis, 100 μl of CM collected
from days 3, 7, 10, and 14 were used for the assay and the
absorbance was read at 450 nm using a Multiskan GO plate
reader (Thermo Scientic, UK). The quantity of soluble
VE-cadherin or VCAM-1 released was deduced by calcu-
lating against their respective standard curves.
2.6.2 qRT-PCR analysis to determine functional gene
expression in endothelialized SDF-1αGAS
Cells from the scaffolds were harvested on the 14th day for
gene expression analysis. RNA extraction and reverse
transcription was performed as described in previous
chapter. qRT-PCR was then performed on cDNA using the
following primers Hs_CXCL12_1_SG, Hs_CXCR4_1_SG,
and Hs_NOS3_1_SG, which encodes for human SDF-1α,
CXCR4, and eNOS, respectively. Fold change in mRNA
expression relative to the untransfected control was calcu-
lated using the 2ΔΔCT method [35] from averages of three
samples for each group. GAPDH (Hs_GAPDH_1_SG) was
used as the housekeeping gene.
2.6.3 Immunouorescent imaging
After 14 days of culture, samples were processed for
immunouorescent imaging. Briey, the samples were rst
washed with PBS and xed in 10% neutral buffered for-
malin for 20 min, and then processed using the standard
protocol for parafnization. The samples were then cut into
8-μm thick slices, deparafnized, and mounted on slides.
Prior to staining, the cells were permeabilized with 0.2%
Tween®20 (Sigma-Aldrich, France) solution in 1x PBS for
30 min (10 min wash × 3) and blocked using 10% normal
goat serum (Invitrogen, UK)/5% BSA/0.3 M glycine (pre-
pared in permeabilizing solution) for 1 h to inhibit non-
specic protein interaction. The slides were then stained
using monoclonal antibodies of CD31 (1:50; ab119339,
Abcam, UK) and SDF-1α(1:100; ab155090, Abcam, UK)
to visualize endothelial morphogenesis and compare the
expression of SDF-1αproteins, respectively.
The next day, the slides were rinsed in PBS thrice for
23 min each to remove any unbound primary antibodies.
Subsequently, the slides were incubated in either Alexa 488-
conjugated goat anti-mouse IgG (A32723, Invitrogen, UK) or
Alexa 594-conjugated goat anti-rabbit IgG (A11012, Invi-
trogen, UK) at 1:800 dilution at room temperature for 1 h in
dark. The rinsing step was performed as before and stained
for nuclei using the mounting medium with DAPI (ab104139,
Abcam, UK) and covered with cover slips. Images were then
captured using uorescence microscope (Olympus BX43,
Japan) at 40× magnication. Samples incubated with only
secondary antibodies were used as controls. All the antibodies
were diluted in 1% BSA in PBS prior to use.
2.6.4 Image analysis
The ImageJsoftware (ImageJ, NIH, Maryland, USA) was
used to semiquantitatively determine the amount of
expressed proteins. For each marker, a constant threshold
value was rst determined through preliminary imaging of
various sections. Using the set threshold value, integrated
density (stained area × mean gray value) of the images was
determined and then normalized to the number of cells
(DAPI counting) to give a nal mean uorescence density
per cell. An average was quantied from four to ve ran-
dom images per sample, with a minimum of three samples
per group. The averages obtained from each group were
then used for statistical comparisons between the groups.
2.6.5 Bioactivity analysis of secreted factors from coculture
on SDF-1αGAS on human SCs
In order to study if the vascularized construct possesses
proneurogenic properties, CM (day 7) collected from the
coculture groups was exposed to human SCs (iXCells Bio-
technologies, USA). Human SCs at passages 3 and 4 were
seeded onto poly-l-lysine (P4707, Sigma, UK) coated 12-
well plates at a density of 30,000 cells/well and allowed to
grow until conuency. Injury was made to the monolayer of
SCs by creating a scratch using a sterile 200 μl pipette tip.
The cells were gently rinsed with PBS to remove any cellular
debris and treated with CM from the vascularized constructs.
Cellular organization was then observed until 48 h. Quanti-
cation of the cellular area was performed using ImageJ from
at least ve images per well (n=3).
2.7 Statistical analysis
All results are expressed as mean ± standard deviation.
Unpaired, two-tailed, t-test was used to demonstrate the
statistical signicance between groups, where p< 0.05 was
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considered to be signicant. One-way ANOVA was also
used to compare statistical difference between the multiple
groups, considering p< 0.05 to be signicant.
3 Results
3.1 PEIpSDF-1αpolyplex effectively transfected
HUVECs and induced the secretion of functional
SDF-1αproteins
MTS assay showed that at days 3 and 7, HUVECs transfected
with PEIpSDF-1αsignicantly promoted their survivability
than the groups transfected with polyplex carrying the non-
therapeutic gene, pLuc (Fig. 1A). Subsequent SDF-1αELISA
analysis showed that protein production reached the max-
imum (565 ± 35 pg/ml) on the 7th day but dropped sig-
nicantly (p< 0.001) at later time points (Fig. 1B). Prior to the
addition of ADSCs, HUVECs secreted comparable levels of
SDF-1α(530 ± 95 pg/ml) as HUVECs monoculture group.
However, SDF-1αprotein levels in the CM were not detect-
able on the days after the HUVECs were cocultured with
ADSCs. Furthermore, on the 14th day, phase-contrast
microscopy revealed that the transfected HUVECs when
cocultured with ADSCs displayed the formation of elongated
cellular clusters with cellular extensions appearing out of the
cluster in multiple directions (Fig. 1C-iv). The untransfected
coculture group also exhibited the formation of interconnected
cellular network. However, cobblestone-like morphological
features of the individual endothelial cells remained distin-
guishable (Fig. 1C-iii). Clearly, morphological differences
between the coculture groups suggest that PEIpSDF-1α
transfected HUVECs secrete functional proteins capable of
recruiting ADSCs for enhanced functional interaction.
3.2 SDF-1αGAS effectively engages endothelial VE-
cadherin while promoting angiogenesis
Angiogenesis is a dynamic process that occurs through
transient disruption of the endothelial adherent junctions
[36]. During the junctional disruption, the adhesion mole-
cules, primarily the VE-cadherin is released as a soluble
form [37]. Therefore, in order to measure angiogenesis
within the SDF-1αGAS, we assayed soluble VE-cadherin
released during the culture period (Fig. 2). All the groups
demonstrated a transient trend in the release of soluble VE-
cadherin that peaked at day 10. Among the groups,
HUVECs on gene-free scaffold released the highest amount
of VE-cadherin at all time points (up to 5.83 ± 0.3 ng/ml at
day 10). When ADSCs were added to the preformed
endothelial network, the release of soluble VE-cadherin was
strongly attenuated. However, we noted a similar amount of
VE-cadherin released by the HUVECs on SDF-1αGAS as
that of the coculture on the gene-free scaffold. Considering
the impact of SDF-1αGAS on HUVECs, we anticipated
more pronounced attenuation of VE-cadherin release in the
coculture on SDF-1αGAS. We conrmed this event by
measuring the level of VE-cadherin from the coculture on
SDF-1αGAS, which showed the lowest (1.27 ± 0.45 ng/ml
at day 10) at all time points. Taken together, it implies that
SDF-1αGAS engages the endothelial VE-cadherin to
modulate angiogenesis in a transient manner.
The release of soluble VE-cadherin could also be mediated
by inammation and its level corresponds to the degree of
inammation [38,39]. Therefore, having observed high level
of soluble VE-cadherin with HUVECs on gene-free scaffold,
we assessed if vascular inammation was involved. This was
conducted by measuring the release of inammation-
associated soluble VCAM-1 [40]. In all the groups, no
detectable amounts of soluble VCAM-1 were released, sup-
porting the notion that the release of VE-cadherin was a
physiological process to promote angiogenesis.
3.3 Coculture enhances SDF-1α-mediated
provasculogenic gene expression
Having noted that cultures on SDF-1αGAS effectively pre-
serve endothelial junctional integrity during angiogenesis, gene
expression was performed at day 14 to determine endothelial
maturation (Fig. 3). First, we measured the levels of SDF-1α
mRNA and noted that coculturing with ADSCs enhances the
expression of SDF-1αmRNA than HUVECs alone. The
coculture on the gene-free scaffold expressed the SDF-1α
mRNA twice as high as HUVECs on the SDF-1αGAS. A
more pronounced expression was noted in the coculture on the
SDF-1αGAS that showed an eightfold higher expression of
the SDF-1αmRNA than its gene-free equivalent.
Provasculogenic maturation was then determined based
on the expression of SDF-1αs downstream effector
mRNAs, namely CXCR4 and eNOS. We noted that the
SDF-1αGAS is not sufciently potent to drive HUVECs
toward a provasculogenic maturation. The SDF-1αGAS
caused a modest increase (43%) in the expression of eNOS
in HUVECs. However, coculturing with ADSCs sig-
nicantly upregulated the expression of eNOS than the
HUVECs on SDF-1αGAS. Furthermore, the coculture on
SDF-1αGAS exhibited a sixfold higher expression of
eNOS mRNA than the HUVECs on the SDF-1αGAS. This
nding suggests that coculture is crucial for enhancing
endothelial maturation in SDF-1αGAS.
3.4 SDF-1αGAS supports endothelial
morphogenesis
Immunostaining with the primary marker for endothelial
cells CD31 revealed that all the groups had undergone
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morphogenesis representative of a capillary-like network
(Fig. 4A). Having conrmed this, SDF-1αwas double
immunostained with CD31 (Fig. 4B, C) to visualize its
spatial distribution on the cellular surface as well as the
level of expression. Mean uorescence density analysis of
SDF-1αrevealed a progressive increase in the expression of
SDF-1αfrom the monocultures of HUVECs toward the
coculture group (Fig. 4D). However, no signicant differ-
ence was noted between HUVECs on the SDF-1αGAS and
the coculture groups. Only the coculture on SDF-1αGAS
exhibited signicantly higher levels of SDF-1αthan the
HUVECs on the gene-free scaffold. Spatially, the SDF-1α
Fig. 1 Effect of PEIpSDF-1αtransfection on HUVECs and its impact
on ADSCs. APEIpSDF-1αtransfected HUVECs exhibited sig-
nicantly enhanced survivability response compared to those trans-
fected nontherapeutic PEIpLuc polyplex. BTransfection with
PEIpSDF-1αinduced transient production of the target protein over a
period of 2 weeks. CPhase-contrast microscopy images of morpho-
logical changes in HUVECs and its coculture with ADSCs at day 14. i
Untransfected HUVECs maintained its cobblestone-like morphology.
ii Transfected HUVECs appeared more polarized. iii Addition of
ADSCs to untransfected HUVECs resulted in the formation of inter-
connected cellular network within the monolayer assembly. iv Elon-
gated, pseudo three-dimensional dense cellular clusters were formed in
transfected coculture group. Data are plotted as mean ± standard
deviation (n=6)
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in HUVECs on SDF-1αGAS appears to be bound to the
cellular surface (indicated by arrows, Fig. 4C-ii). On the
other hand, SDF-1αin coculture on SDF-1αGAS appears
to be secreted into the extracellular space (indicated by
arrows, Fig. 4C-iv). Overall, this nding demonstrates that
SDF-1αGAS effectively supports angiogenesis and that
coculture enhances SDF-1αprotein expression.
3.5 Coculture on SDF-1αGAS induces human SCs
morphogenesis
In addition to vasculogenesis, neuronal regeneration is an
important aspect of wound healing that is often over-
looked. During angiogenesis, the angiogenic vessels guide
the SCs to the site of wound [31]. The SCs then lay down
matrix to guide regenerating axons and promote their
reinnervation of the epidermis [32]. Therefore, having
observed enhanced vasculogenic response in the coculture
groups, the potential of the vascularized constructs to
signal SCs was investigated.
Twenty-four hours post exposure to CM, the SCs
actively invaded the wound zone regardless of the source of
the CM (Fig. 5B). However, further incubation of the SCs
in the CM up to 48 h resulted in the generation of cluster-
like structures (Fig. 5C). These clusters formed connections
between other clusters through slender cord-like structures
that protruded out of each clusters. The clusters did not have
dened boundaries however measurement of the total spa-
tial coverage revealed that the CM from coculture on SDF-
1αGAS generated signicantly larger SCs clusters than the
CM from its gene-free equivalent (Fig. 5C-ii). In addition,
the cord-like structures connecting the large clusters were
highly aligned and more parallelly arranged than the con-
nections between the smaller clusters (Fig. 5C-ii). From
these observations, it is evident that vascularized SDF-1α
GAS produces signaling components that can stimulate
proneurogenic behavior in SCs implicated for neuronal
regeneration.
4 Discussion
The overall objective of the study was to assess the bioac-
tivity of SDF-1αGAS on human endothelial cells and its
coculture with human ADSCs. A preliminary 2D study
showed that the HUVECs transfected with PEIpSDF-1α
polyplex transiently secreted SDF-1αproteins that actively
recruited ADSCs and enhanced their adhesion to the
HUVECs. On the SDF-1αGAS, the HUVECs displayed
angiogenesis that occurred in a transient manner through
regulation of the junctional adhesion molecule VE-
cadherin. Coculturing with ADSCs signicantly improved
the preservation of the VE-cadherin and also enhanced the
maturation of endothelial cells through the expression of
downstream effectors CXCR4 and eNOS. Secreted factors
from the provasculogenic vessels on SDF-1αGAS further
displayed strong bioactive properties toward human SCs
that organized into Bünger band-like structures. Together,
this study showed that SDF-1αGAS is a highly functional
biomaterial scaffold capable of enhancing provasculogenic
response for wound healing applications.
In the application of synthetic gene-delivery vectors, the
survivability of the cells post transfection is critical for
offering the optimal therapeutic response [4143]. There-
fore, we rst evaluated the safety of the polyplexes on the
HUVECs. First, we noted a proliferation curve that peaked
at day 3. This appears to be a general manner of pro-
liferation of HUVECs in EGM, as a similar case has been
observed in other studies, which validated the behavior
through the measurement of proliferation marker Ki67 [44].
Nevertheless, our nding indicates that despite an initial
insult to HUVECshealth due to the exposure to poly-
plexes, the delivery of SDF-1αgene signicantly enhanced
the survivability of HUVECs than the ones transfected with
Luc gene (Fig. 1A). This nding possibly suggests that
secreted SDF-1αproteins may have exerted a protective
effect on the HUVECs itself, mediated by a positive auto-
crine loop [45]. Our analysis of SDF-1αprotein production
further proved that the transfected HUVECs indeed pro-
duced high levels of SDF-1αprotein that lasted up to
14 days (Fig. 1B). We further conrmed that SDF-1αpro-
tein was also highly bioactive toward the ADSCs as a
greater clustering of the cells occurred in the coculture
Fig. 2 Temporal regulation of soluble VE-cadherin from endothelia-
lized gene-free scaffold and SDF-1αGAS. SDF-1αGAS strongly
affects the vascular growth of endothelial cells by suppressing the
release of soluble VE-cadherin. Coculturing with ADSCs offers further
control on the release of soluble VE-cadherin from endothelial cells.
HUVECs on SDF-1αGAS demonstrated signicant reduction in the
levels of soluble VE-cadherin at days 7 (p< 0.05) and 10 (p< 0.0005)
relative to HUVECs on gene-free scaffold. Coculture on SDF-1αGAS
strongly attenuated the release of VE-cadherin at all time points. Data
are presented as mean ± standard deviation. One-way ANOVA was
used to deduce statistical signicance. *, **, ***, and **** indicate
statistical signicance of p< 0.05, p< 0.01, p< 0.005, and p< 0.0005,
respectively
Journal of Materials Science: Materials in Medicine (2021) 32:26 Page 7 of 13 26
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
group consisting of SDF-1αproducing HUVECs. The
response to SDF-1αis potentially mediated by CXCR4 in
ADSCs that play a specic role in SDF-1αdriven chemo-
taxis [46].
Normally, to measure angiogenesis on 3D scaffolds over
time, the endothelialized constructs are harvested at pre-
specied time points and imaged for capillary-like network
formation [14,25]. Here, we show a relatively simpler
alternative method that allows continuous monitoring of the
endothelial growth in a 3D scaffold without the need for
harvesting the cellularized samples. This technique also
offers another advantage by providing information of the
dynamic changes in endothelial junctional stability, which
is crucial for endothelial homeostasis [36,47]. Based on a
previous study [37], which described that the endothelial
junctional adhesion molecule VE-cadherin is proteolytically
cleaved during angiogenesis and shed as a soluble form, we
measured the amount of VE-cadherin released from the
endothelialized constructs. The HUVECs on the gene-free
scaffold showed a clear transient release of the soluble VE-
cadherin that peaked at day 10. This nding conrms that
the VE-cadherin is temporally involved in endothelial
angiogenesis. Furthermore, the pattern of VE-cadherin
release is also in agreement with ndings of both in vitro
[14,25] and in vivo [48] studies, which showed that the
angiogenesis peaks between days 7 and 14. Moreover,
comparing the levels of VE-cadherin revealed a potential
role of SDF-1αGAS that it may be involved in the
enhancement of homophilic endothelial-to-endothelial
adhesion mediated by VE-cadherin [49]. This speculation
is derived from the nding that the HUVECs on SDF-1α
GAS released similar levels of VE-cadherin as that of the
coculture on the gene-free scaffold. The cocultures are
generally known to enhance the expression of cellular VE-
cadherin and engage them in the restoration of endothelial
barrier functions [5052].
Fig. 3 Gene expression analysis of HUVECs and its coculture on
gene-free scaffolds and SDF-1αGAS. SDF-1αGAS signicantly
increased the expression of mRNAs for SDF-1αand its cognate
receptor CXCR4 in HUVECs. Coculture with ADSCs signicantly
elevated the expression of downstream effector genes of SDF-1α
CXCR4 and eNOS than the HUVECs on SDF-1αGAS. Data are
plotted as mean ± standard deviation (n=3). *, **, and *** indicate
statistical signicance of p< 0.05, p< 0.01, and p< 0.005, respectively
26 Page 8 of 13 Journal of Materials Science: Materials in Medicine (2021) 32:26
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
The signicant activation of CXCR4 in the HUVECs on
SDF-1αGAS presents a potential mechanism that may have
controlled the release of VE-cadherin from the HUVECs. The
SDF-1α-induced CXCR4 may sustain the expression of VE-
cadherin and maintain the endothelial barrier functions via the
activation of Wnt/β-catenin pathway [53]. The stem cells are
alsoknowntoemploytheβ-cateninpathwaytoenhancethe
VE-cadherin-mediated barrier properties of endothelial cells
[51]. Importantly, gene expression analysis further showed
that the SDF-1αGAS alone is insufcient to drive pro-
vasculogenic response in HUVECs and that coculturing with
ADSCs was crucial for enhancement of the provasculogenic
gene eNOS. Similar enhancement in the expression of eNOS
mRNA in the coculture of endothelial cells and ADSCs has
Fig. 4 Visualization of
endothelial anastomosis and
SDF-1αexpression. AAll the
cultures showed strong
immunoreactivity to CD31 and
exhibited endothelial
morphogenesis representative of
capillary-like network.
BHUVECs on the gene-free
scaffold expressed the lowest
amount of SDF-1αproteins.
CMagnied images of the
endothelial network showing the
differences in spatial distribution
of SDF-1αbetween the groups.
DQuantitative representation of
SDF-1αprotein expression
showing an increasing trend
toward the coculture group. Data
are presented as mean ± standard
deviation. * indicates statistical
signicance of p< 0.05
Journal of Materials Science: Materials in Medicine (2021) 32:26 Page 9 of 13 26
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
also been observed in other studies [54]. Although we
investigated this study as an approach for enhancing blood
vessel regeneration in a biomaterial scaffold, SDF-1αGAS
may also be useful for promoting lymphangiogenesis. The
lymphatic vessels regulate immune cell trafcking and tissue
uid homeostasis [55]. In addition to angiogenesis, lym-
phangiogenesis also actively participates in the wound healing
process and relies on the growth of lymphatic endothelial cells
(LECs) [56]. VEGF-C is a potent stimulant of LECs [56].
However, similar to angiogenesis, studies have found that the
interaction of LECs with stem cells is crucial for expanding
the lymphatic endothelial network [57,58]. Moreover, the
ADSCs, despite the low production of VEGF-C, were
found to enhance the growth of LECs effectively [59].
Fig. 5 Cellular organization of
human Schwann cells in
response to CM derived from
coculture of endothelial cells
and ADSCs. AA scratched
monolayer of Schwann cells.
BSchwann cells invading the
wounded zone 24 h post
exposure to CM. CSchwann
cells undergoing morphological
changes 48 h post exposure to
CM. DAt 48 h, Schwann cells
exposed to CM from the
coculture on SDF-1αGAS
organized into signicantly (p<
0.01) larger interconnected
clusters than the Schwann cells
exposed to CM from the
coculture on gene-free scaffold.
Data are presented as mean ±
standard deviation
26 Page 10 of 13 Journal of Materials Science: Materials in Medicine (2021) 32:26
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Therefore, given the importance of endothelial cellsinterac-
tion with stem cells for lymphangiogenesis, the ability of
SDF-1αGAS to induce the overexpression of chemotactic
SDF-1αmay facilitate improved intercellular interaction and
enhance functional growth.
In order to readdress the importance of neuronal activa-
tion while considering strategies for wound healing, we
ultimately investigated if the provasculogenic coculture
constructs can signal SCs. SCs were specically chosen
because, in the process of reinnervation, SCs invasion
precedes axonal regeneration [60,61]. During angiogenesis,
invading blood vessels guide the SCs into the wound site
[31,61]. The SCs then lay down matrix and guide the
regenerating axons for epidermal reinnervation [61].
We validated that the CM produced by the coculture on
the SDF-1αGAS possessed superior bioactive properties
than its gene-free scaffold equivalent. However, an apparent
difference in the response of SCs appeared only at 48 h post
exposure to CM. The SCs exposed to CM from the SDF-1α
GAS group grew into signicantly large clusters. Clusters
incubated in the CM from the SDF-1αGAS group also
developed parallelly arranged cord-like structures connect-
ing other clusters. This cellular arrangement has also been
demonstrated by SCs when they were cocultured with
neural cells such as the meningeal cells [62,63]. These
cellular arrangement are presumed a transformation of the
SCs into Bünger bands, which is necessary for supporting
axonal regeneration across wound [32,62].
To this end, we show that the provasculogenic structures
created on SDF-1αGAS are highly potent bioactive con-
structs for wound healing applications. Nevertheless, it
would be interesting to see how the SDF-1αGAS might
work on adult endothelial cells such as the peripheral blood
or umbilical cord blood-derived endothelial cells (ECFCs)
[64] and the induced pluripotent stem cells derived endo-
thelial cells (iPSCs-ECs) [65]. Both ECFCs and iPSCs are
known to show high proangiogenic activity both in vitro
[66] and in vivo [64,65]. Moreover, ECFCs are also known
to possess stem cell-like properties [67], which may offer
enhanced therapeutic effects. These cell types are of parti-
cular interest as they can be harvested from the patient and
representing clinically translatable cell candidates for
autologous therapy [68,69]. In the future, we will further
attempt to investigate the efcacy of the prevascularized
construct in vivo, which is currently a limitation of the
study, using established techniques such as subcutaneous
implantation in nude mice.
5 Conclusion
This study explored the functional impact of SDF-1αGAS
on human endothelial cells and its coculture with human
ADSCs. Particularly, we have shown that SDF-1αGAS
effectively supports endothelial angiogenesis by desirably
controlling the junctional adhesive effect, which is essential
for maturation of the endothelial network. Better maturation
could be achieved through coculturing with ADSCs, which
was further enhanced when supported with SDF-1αGAS.
In addition, SDF-1αGAS activated coculture group
demonstrated a strong capacity to signal SCs to differentiate
toward a proneurogenic phenotype by inuencing their
cellular organization into Bünger band-like structures.
Taken together, we showed a potent bioinstructive role of
SDF-1αGAS for the generation of bioactive provasculo-
genic constructs for enhanced wound healing applications.
Acknowledgements This work was funded by RCSI-Dilmun PhD
scholarship.
Compliance with ethical standards
Conict of interest The authors declare no competing interests.
Publishers note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
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long as you give appropriate credit to the original author(s) and the
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Ensuring an adequate angiogenic response during wound healing is a prevailing clinical challenge in biomaterials science. To address this, we aimed to develop a pro-angiogenic gene-activated scaffold (GAS) that could activate MSCs to produce paracrine factors and influence angiogenesis and wound repair. A non-viral polyethyleneimine (PEI) nanoparticles carrying a gene encoding for stromal derived factor-1 alpha (SDF-1α) was combined with a collagen-chondroitin sulfate scaffold to produce the GAS. The ability of this platform to enhance the angiogenic potential of mesenchymal stem cells (MSCs) was then assessed. We found that the MSCs on GAS exhibited early over-expression of SDF-1α mRNA with the activation of angiogenic markers VEGF and CXCR4. Exposing endothelial cells to conditioned media collected from GAS supported MSCs promoted a 20% increase in viability and 33% increase in tubule formation (p<0.05). Furthermore, the conditioned media promoted a 50% increase in endothelial cell migration and wound closure (p<0.005). Gene expression analysis of the endothelial cells revealed that the functional response was associated with up-regulation of angiogenic genes; VEGF, CXCR4, eNOS and SDF-1α. Overall, this study shows collagen-based scaffolds combined with SDF-1α gene therapy can provide enhanced pro-angiogenic response, suggesting a promising approach to overcome poor vasculature during wound healing.