ArticlePDF Available

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


Abstract and Figures

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.
This content is subject to copyright. Terms and conditions apply.
Journal of Materials Science: Materials in Medicine (2021) 32:26
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
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
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
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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
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)
26 Page 2 of 13 Journal of Materials Science: Materials in Medicine (2021) 32:26
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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
Journal of Materials Science: Materials in Medicine (2021) 32:26 Page 3 of 13 26
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
26 Page 4 of 13 Journal of Materials Science: Materials in Medicine (2021) 32:26
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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
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
Immunostaining with the primary marker for endothelial
cells CD31 revealed that all the groups had undergone
Journal of Materials Science: Materials in Medicine (2021) 32:26 Page 5 of 13 26
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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)
26 Page 6 of 13 Journal of Materials Science: Materials in Medicine (2021) 32:26
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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
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
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,
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
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
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,
adaptation, distribution and reproduction in any medium or format, as
long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons license, and indicate if
changes were made. The images or other third party material in this
article are included in the articles Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not
included in the articles Creative Commons license and your intended
use is not permitted by statutory regulation or exceeds the permitted
use, you will need to obtain permission directly from the copyright
holder. To view a copy of this license, visit http://creativecommons.
1. West JL, Moon JJ. Vascularization of engineered tissues:
approaches to promote angiogenesis in biomaterials. Curr Top
Med Chem. 2008;8:30010.
2. Quinlan E, LópezNoriega A, Thompson EM, Hibbitts A, Cryan SA,
OBrien FJ. Controlled release of vascular endothelial growth factor
from spraydried alginate microparticles in collagenhydroxyapatite
scaffolds for promoting vascularization and bone repair. J Tissue Eng
Regen Med. 2017;11:1097109.
3. Losi P, Briganti E, Errico C, Lisella A, Sanguinetti E, Chiellini F,
et al. Fibrin-based scaffold incorporating VEGF-and bFGF-loaded
nanoparticles stimulates wound healing in diabetic mice. Acta
Biomater. 2013;9:781421.
4. Kanda N, Morimoto N, Ayvazyan AA, Takemoto S, Kawai K,
Nakamura Y, et al. Evaluation of a novel collagengelatin scaffold
for achieving the sustained release of basic broblast growth factor
in a diabetic mouse model. J Tissue Eng Regen Med. 2014;8:2940.
5. Laiva AL, OBrien FJ, Keogh MB. Innovations in gene and
growth factor delivery systems for diabetic wound healing. J
Tissue Eng Regen Med. 2018;12:e296312.
Journal of Materials Science: Materials in Medicine (2021) 32:26 Page 11 of 13 26
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
6. Davies NH, Schmidt C, Bezuidenhout D, Zilla P. Sustaining
neovascularization of a scaffold through staged release of vascular
endothelial growth factor-A and platelet-derived growth factor-
BB. Tissue Eng Part A. 2011;18:2634.
7. Wang Z, Wang Z, Lu WW, Zhen W, Yang D, Peng S. Novel
biomaterial strategies for controlled growth factor delivery for
biomedical applications. NPG Asia Mater. 2017;9:e435.
8. Lloyd-Grifth C, McFadden TM, Duffy GP, Unger RE,
Kirkpatrick CJ, OBrien FJ. The pre-vascularisation of a collagen-
chondroitin sulphate scaffold using human amniotic uid-derived
stem cells to enhance and stabilise endothelial cell-mediated
vessel formation. Acta Biomater. 2015;26:26373.
9. Duffy GP, McFadden TM, Byrne EM, Gill S-L, Farrell E, OBrien
FJ. Towards in vitro vascularisation of collagen-GAG scaffolds.
Eur Cell Mater. 2011;21:e30.
10. McFadden T, Duffy G, Allen A, Stevens H, Schwarzmaier S,
Plesnila N, et al. The delayed addition of human mesenchymal
stem cells to pre-formed endothelial cell networks results in
functional vascularization of a collagenglycosaminoglycan
scaffold in vivo. Acta Biomater. 2013;9:930316.
11. Zhang W, Wray LS, Rnjak-Kovacina J, Xu L, Zou D, Wang S,
et al. Vascularization of hollow channel-modied porous silk
scaffolds with endothelial cells for tissue regeneration. Bioma-
terials. 2015;56:6877.
12. Unger RE, Ghanaati S, Orth C, Sartoris A, Barbeck M, Halstenberg S,
et al. The rapid anastomosis between prevascularized networks on silk
broin scaffolds generated in vitro with cocultures of human micro-
vascular endothelial and osteoblast cells and the host vasculature.
Biomaterials. 2010;31:695967.
13. Kress S, Baur J, Otto C, Burkard N, Braspenning J, Walles H,
et al. Evaluation of a miniaturized biologically vascularized
scaffold in vitro and in vivo. Sci Rep. 2018;8:4719.
14. do Amaral RJFC, Cavanagh B, OBrien FJ, Kearney CJ. Plate-
letderived growth factor stabilises vascularisation in
collagenglycosaminoglycan scaffolds in vitro. J Tissue Eng
Regen Med. 2019;13:26173.
15. Raftery RM, Castaño IM, Chen G, Cavanagh B, Quinn B, Curtin
CM, et al. Translating the role of osteogenic-angiogenic coupling
in bone formation: Highly efcient chitosan-pDNA activated
scaffolds can accelerate bone regeneration in critical-sized bone
defects. Biomaterials. 2017;149:11627.
16. Curtin CM, Tierney EG, McSorley K, Cryan SA, Duffy GP,
OBrien FJ. Combinatorial gene therapy accelerates bone
regeneration: nonviral dual delivery of VEGF and BMP2 in a
collagennanohydroxyapatite scaffold. Adv Health Mater.
17. Raftery RM, Walsh DP, Ferreras LB, Castaño IM, Chen G,
LeMoine M, et al. Highly versatile cell-penetrating peptide loaded
scaffold for efcient and localized gene delivery to multiple cell
types: from development to application in tissue engineering.
Biomaterials. 2019;216:119277.
18. Lackington WA, Raftery RM, OBrien FJ. In vitro efcacy of a
gene-activated nerve guidance conduit incorporating non-viral
PEI-pDNA nanoparticles carrying genes encoding for NGF,
GDNF and c-Jun. Acta Biomater. 2018;75:11528.
19. Tierney EG, Duffy GP, Hibbitts AJ, Cryan S-A, OBrien FJ. The
development of non-viral gene-activated matrices for bone
regeneration using polyethyleneimine (PEI) and collagen-based
scaffolds. J Control Release. 2012;158:30411.
20. Laiva AL, Raftery RM, Keogh MB, Obrien FJ. Pro-angiogenic
impact of SDF-1αgene-activated collagen-based scaffolds in stem
cell driven angiogenesis. Int J Pharm. 2018;544:3729.
21. Gallagher KA, Liu Z-J, Xiao M, Chen H, Goldstein LJ, Buerk
DG, et al. Diabetic impairments in NO-mediated endothelial
progenitor cell mobilization and homing are reversed by hyper-
oxia and SDF-1α. J Clin Investig. 2007;117:124959.
22. Yu J, Sievers RE, Lee RJ. Effects of scaffold-delivered SDF-1
alpha protein in chronic rat myocardial infarction model. J Med
Biol Eng. 2014;34:2249.
23. Rabbany SY, Pastore J, Yamamoto M, Miller T, Rai S, Aras R,
et al. Continuous delivery of stromal cell-derived factor-1 from
alginate scaffolds accelerates wound healing. Cell Transplant.
24. Kuraitis D, Zhang P, Zhang Y, Padavan D, McEwan K,
Sofrenovic T, et al. A stromal cell-derived factor-1 releasing matrix
enhances the progenitor cell response and blood vessel growth in
ischaemic skeletal muscle. Eur Cell Mater. 2011;22:e23.
25. Freiman A, Shandalov Y, Rozenfeld D, Shor E, Segal S, Ben-
David D, et al. Adipose-derived endothelial and mesenchymal
stem cells enhance vascular network formation on three-
dimensional constructs in vitro. Stem Cell Res Ther. 2016;7:5.
26. Liu S, Zhang H, Zhang X, Lu W, Huang X, Xie H, et al.
Synergistic angiogenesis promoting effects of extracellular matrix
scaffolds and adipose-derived stem cells during wound repair.
Tissue Eng Part A. 2010;17:72539.
27. Mazzocchi AR, Man AJ, DesOrmeaux J-PS, Gaborski TR. Porous
membranes promote endothelial differentiation of adipose-derived
stem cells and perivascular interactions. Cell Mol Bioeng.
28. Deng M, Gu Y, Liu Z, Qi Y, Ma GE, Kang N. Endothelial dif-
ferentiation of human adipose-derived stem cells on polyglycolic
acid/polylactic acid mesh. Stem Cells Int. 2015;2015:111.
29. Jabbarzadeh E, Starnes T, Khan YM, Jiang T, Wirtel AJ, Deng M,
et al. Induction of angiogenesis in tissue-engineered scaffolds
designed for bone repair: a combined gene therapycell transplan-
tation approach. Proc Natl Acad Sci USA. 2008;105:11099104.
30. Jessen K, Mirsky R. The repair Schwann cell and its function in
regenerating nerves. J Physiol. 2016;594:352131.
31. Cattin A-L, Burden JJ, Van Emmenis L, Mackenzie FE, Hoving
JJ, Calavia NG, et al. Macrophage-induced blood vessels guide
Schwann cell-mediated regeneration of peripheral nerves. Cell.
32. Parrinello S, Napoli I, Ribeiro S, Digby PW, Fedorova M,
Parkinson DB, et al. EphB signaling directs peripheral nerve
regeneration through Sox2-dependent Schwann cell sorting. Cell.
33. Ramos T, Ahmed M, Wieringa P, Moroni L. Schwann cells pro-
mote endothelial cell migration. Cell Adhes Migr. 2015;9:44151.
34. Haugh MG, Murphy CM, OBrien FJ. Novel freeze-drying
methods to produce a range of collagenglycosaminoglycan
scaffolds with tailored mean pore sizes. Tissue Eng Part C:
Methods. 2009;16:88794.
35. Livak KJ, Schmittgen TD. Analysis of relative gene expression
data using real-time quantitative PCR and the 2ΔΔCT method.
Methods 2001;25:4028.
36. Di Lorenzo A, Lin MI, Murata T, Landskroner-Eiger S, Schleicher
M, Kothiya M, et al. eNOS-derived nitric oxide regulates endo-
thelial barrier function through VE-cadherin and Rho GTPases. J
Cell Sci. 2013;126:554152.
37. Schulz B, Pruessmeyer J, Maretzky T, Ludwig A, Blobel CP,
Saftig P, et al. ADAM10 regulates endothelial permeability and T-
cell transmigration by proteolysis of vascular endothelial cadherin.
Circ Res. 2008;102:1192201.
38. Sidibé A, Mannic T, Arboleas M, Subileau M, GulinoDebrac D,
Bouillet L, et al. Soluble VEcadherin in rheumatoid arthritis
patients correlates with disease activity: evidence for tumor
necrosis factor αinduced VEcadherin cleavage. Arthritis Rheum.
39. Flemming S, Burkard N, Renschler M, Vielmuth F, Meir M,
Schick MA, et al. Soluble VE-cadherin is involved in endothelial
barrier breakdown in systemic inammation and sepsis. Cardio-
vasc Res. 2015;107:3244.
26 Page 12 of 13 Journal of Materials Science: Materials in Medicine (2021) 32:26
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
40. Garton KJ, Gough PJ, Philalay J, Wille PT, Blobel CP, Whitehead
RH, et al. Stimulated shedding of vascular cell adhesion molecule
1 (VCAM-1) is mediated by tumor necrosis factor-α-converting
enzyme (ADAM 17). J Biol Chem. 2003;278:3745964.
41. Ahn HH, Lee JH, Kim KS, Lee JY, Kim MS, Khang G, et al.
Polyethyleneimine-mediated gene delivery into human adipose
derived stem cells. Biomaterials. 2008;29:241522.
42. Hunt MA, Currie MJ, Robinson BA, Dachs GU. Optimizing
transfection of primary human umbilical vein endothelial cells
using commercially available chemical transfection reagents. J
Biomol Tech. 2010;21:66.
43. Forcato D, Fili A, Alustiza F, Martínez JL, Abel SB, Nicotra MO,
et al. Transfection of bovine fetal broblast with polyethylenimine
(PEI) nanoparticles: effect of particle size and presence of fetal
bovine serum on transgene delivery and cytotoxicity. Cyto-
technology. 2017;69:65565.
44. Gharaei M, Xue Y, Mustafa K, Lie S, Fristad I. Human dental
pulp stromal cell conditioned medium alters endothelial cell
behavior. Stem Cell Res Ther. 2018;9:69.
45. Cho S-W, Yang F, Son SM, Park H-J, Green JJ, Bogatyrev S,
et al. Therapeutic angiogenesis using genetically engineered
human endothelial cells. J Control Release. 2012;160:51524.
46. Li Q, Zhang A, Tao C, Li X, Jin P. The role of SDF-1-CXCR4/
CXCR7 axis in biological behaviors of adipose tissue-derived
mesenchymal stem cells in vitro. Biochem Biophys Res Commun.
47. Giannotta M, Trani M, Dejana E. VE-cadherin and endothelial
adherens junctions: active guardians of vascular integrity. Dev
Cell. 2013;26:44154.
48. Shaterian A, Borboa A, Sawada R, Costantini T, Potenza B,
Coimbra R, et al. Real-time analysis of the kinetics of angiogen-
esis and vascular permeability in an animal model of wound
healing. Burns. 2009;35:8117.
49. Dejana E, Orsenigo F, Lampugnani MG. The role of adherens
junctions and VE-cadherin in the control of vascular permeability.
J Cell Sci. 2008;121:211522.
50. Grainger SJ, Putnam AJ. Assessing the permeability of engineered
capillary networks in a 3D culture. PLoS ONE. 2011;6:e22086.
51. Pati S, Khakoo AY, Zhao J, Jimenez F, Gerber MH, Harting M,
et al. Human mesenchymal stem cells inhibit vascular perme-
ability by modulating vascular endothelial cadherin/β-catenin
signaling. Stem Cells Dev. 2010;20:89101.
52. Chen Q-H, Liu A-R, Qiu H-B, Yang Y. Interaction between
mesenchymal stem cells and endothelial cells restores endothelial
permeability via paracrine hepatocyte growth factor in vitro. Stem
Cell Res Ther. 2015;6:44.
53. Döring Y, Noels H, van der Vorst EP, Neideck C, Egea V,
Drechsler M, et al. Vascular CXCR4 limits atherosclerosis by
maintaining arterial integrity: evidence from mouse and human
studies. Circulation. 2017;136:388403.
54. Gao P, Yang X, Mungur L, Kampo S, Wen Q. Adipose tissue-
derived stem cells attenuate acute lung injury through eNOS and
eNOS-derived NO. Int J Mol Med. 2013;31:13138.
55. Tammela T, Alitalo K. Lymphangiogenesis: molecular mechan-
isms and future promise. Cell. 2010;140:46076.
56. Güç E, Briquez PS, Foretay D, Fankhauser MA, Hubbell JA,
Kilarski WW, et al. Local induction of lymphangiogenesis with
engineered brin-binding VEGF-C promotes wound healing by
increasing immune cell trafcking and matrix remodeling. Bio-
materials. 2017;131:16075.
57. Saijo H, Suzuki K, Yoshimoto H, Imamura Y, Yamashita S,
Tanaka K. Paracrine effects of adipose-derived stem cells promote
lymphangiogenesis in irradiated lymphatic endothelial cells. Plast
Reconstr Surg. 2019;143:1189e200e.
58. Knezevic L, Schaupper M, Mühleder S, Schimek K, Hasenberg T,
Marx U, et al. Engineering blood and lymphatic microvascular
networks in brin matrices. Front Bioeng Biotechnol. 2017;5:25.
59. Ahmadzadeh N, Robering JW, Kengelbach-Weigand A, Al-
Abboodi M, Beier JP, Horch RE, et al. Human adipose-derived
stem cells support lymphangiogenesis in vitro by secretion of
lymphangiogenic factors. Exp Cell Res. 2020;388:111816.
60. Gingras M, Paradis I, Berthod F. Nerve regeneration in a
collagenchitosan tissue-engineered skin transplanted on nude
mice. Biomaterials. 2003;24:165361.
61. Rajan B, Polydefkis M, Hauer P, Grifn JW, McArthur JC.
Epidermal reinnervation after intracutaneous axotomy in man. J
Comp Neurol. 2003;457:2436.
62. Franssen EH, Roet KC, de Bree FM, Verhaagen J. Olfactory
ensheathing glia and Schwann cells exhibit a distinct interaction
behavior with meningeal cells. J Neurosci Res. 2009;87:155664.
63. Roet KC, Wirz KT, Franssen EH, Verhaagen J. A role for neu-
ropilins in the interaction between Schwann cells and meningeal
cells. PLoS ONE. 2014;9:e109401.
64. Au P, Daheron LM, Duda DG, Cohen KS, Tyrrell JA, Lanning
RM, et al. Differential in vivo potential of endothelial progenitor
cells from human umbilical cord blood and adult peripheral blood
to form functional long-lasting vessels. Blood. 2008;111:13025.
65. Clayton ZE, Tan RP, Miravet MM, Lennartsson K, Cooke JP,
Bursill CA, et al. Induced pluripotent stem cell-derived endo-
thelial cells promote angiogenesis and accelerate wound closure
in a murine excisional wound healing model. Biosci Rep.
66. Belair DG, Whisler JA, Valdez J, Velazquez J, Molenda JA,
Vickerman V, et al. Human vascular tissue models formed from
human induced pluripotent stem cell derived endothelial cells.
Stem Cell Rev Rep. 2015;11:51125.
67. Guillevic O, Ferratge S, Pascaud J, Driancourt C, Boyer-Di-Ponio
J, Uzan G. A novel molecular and functional stemness signature
assessing human cord blood-derived endothelial progenitor cell
immaturity. PLoS ONE. 2016;11:e0152993.
68. Paschalaki KE, Randi AM. Recent advances in endothelial colony
forming cells toward their use in clinical translation. Front Med.
69. Haake K, Ackermann M, Lachmann N. Concise review: towards the
clinical translation of induced pluripotent stem cellderived blood
cellsready for takeoff. Stem Cells Transl Med. 2019;8:3329.
Journal of Materials Science: Materials in Medicine (2021) 32:26 Page 13 of 13 26
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
... The lymphangiogenic function increases with infiltrating macrophages that produce (VEGF-C) [35]. In addition to lymphangiogenesis, VEGF-C may regulate physiological angiogenesis [35,36]. Studies have also shown that SIM can suppress the growth of bacterial pathogens [37,38]. ...
Full-text available
Since wound dressing has been considered a promising strategy to improve wound healing, recent attention has been focused on the development of modern wound dressings based on synthetic and bioactive polymers. In this study, we prepared a multifunctional wound dressing based on carboxymethyl chitosan/sodium alginate hydrogel containing a nanostructured lipid carrier in which simvastatin has been encapsulated. This dressing aimed to act as a barrier against pathogens, eliminate excess exudates, and accelerate wound healing by increasing the production of vascular endothelial growth factor (VEGF). Among various fabricated composites of dressing, the hydrogel composite with a carboxymethyl chitosan/sodium alginate ratio of 1:2 had an average pore size of about 98.44 ± 26.9 μm and showed 707 ± 31.9 % swelling and a 2116 ± 79.2 g/m2.per day water vapor transfer rate (WVTR), demonstrating appropriate properties for absorbing exudates and maintaining wound moisture. The nanostructured lipid carrier with optimum composition and properties had a spherical shape and uniform particle size distribution (74.46 ±7.9 nm). The prepared nanocomposite hydrogel displayed excellent antibacterial activity against Escherichia coli and Staphylococcus aureus bacteria as well as high biocompatibility on L929 mouse fibroblast cells. It can release the loaded simvastatin drug slowly and over a prolonged period of time. The highest drug release occurred (80%) within 14 days. The results showed that this novel nanocomposite could be a promising candidate as a wound dressing for treating various chronic wounds in skin tissues.
Full-text available
Wound healing requires a tight orchestration of complex cellular events. Disruption in the cell-signaling events can severely impair healing. The application of biomaterial scaffolds has shown healing potential; however, the potential is insufficient for optimal wound maturation. This study explored the functional impact of a collagen-chondroitin sulfate scaffold functionalized with nanoparticles carrying an anti-aging gene β-Klotho on human adipose-derived stem cells (ADSCs) for rejuvenative healing applications. We studied the response in the ADSCs in three phases: (1) transcriptional activities of pluripotency factors (Oct-4, Nanog and Sox-2), proliferation marker (Ki-67), wound healing regulators (TGF-β3 and TGF-β1); (2) paracrine bioactivity of the secretome generated by the ADSCs; and (3) regeneration of basement membrane (fibronectin, laminin, and collagen IV proteins) and expression of scar-associated proteins (α-SMA and elastin proteins) towards maturation. Overall, we found that the β-Klotho gene-activated scaffold offers controlled activation of ADSCs’ regenerative abilities. On day 3, the ADSCs on the gene-activated scaffold showed enhanced (2.5-fold) activation of transcription factor Oct-4 that was regulated transiently. This response was accompanied by a 3.6-fold increase in the expression of the anti-fibrotic gene TGF-β3. Through paracrine signaling, the ADSCs-laden gene-activated scaffold also controlled human endothelial angiogenesis and pro-fibrotic response in dermal fibroblasts. Towards maturation, the ADSCs-laden gene-activated scaffold further showed an enhanced regeneration of the basement membrane through increases in laminin (2.1-fold) and collagen IV (8.8-fold) deposition. The ADSCs also expressed 2-fold lower amounts of the scar-associated α-SMA protein with improved qualitative elastin matrix deposition. Collectively, we determined that the β-Klotho gene-activated scaffold possesses tremendous potential for wound healing and could advance stem cell-based therapy for rejuvenative healing applications.
Full-text available
Background: There is currently no reliable treatment for secondary lymphedema caused by lymph node dissection or radiotherapy; however, stem cell-based regenerative medicine is emerging as a promising remedy for such complications. Adipose-derived stem cells (ADSC) have recently attracted attention as they are easily accessible. The purpose of this study was to examine the effects of ADSC on lymphangiogenesis involving human dermal lymphatic endothelial cells (HDLEC) exposed to ionizing radiation. Methods: Proliferation, migration, and tube-formation were analyzed in HDLEC that were co-cultured with ADSC or cultured in ADSC-conditioned medium. The levels of lymphangiogenic factors secreted from ADSC were analyzed by Enzyme-linked immunosorbent assays (ELISA) and Western blotting. Results: Co-culturing with ADSC and the use of ADSC-conditioned medium both significantly promoted proliferation, migration, and tube-formation in non-irradiated HDLEC. We also found that irradiated ADSC had similar alleviative effects on irradiated HDLEC. ELISA and Western blotting analysis revealed that irradiating ADSC increased their secretion of basic fibroblast growth factor (bFGF) in a dose-dependent manner, while it caused no detectable change in their secretion of vascular endothelial growth factor (VEGF)-A, VEGF-C, or hepatocyte growth factor. Conclusion: These results demonstrated that factors secreted by ADSC contribute to the promotion of lymphangiogenesis in irradiated HDLEC. Our findings also suggest that radiation potentiates the paracrine effects of ADSC by stimulating bFGF protein expression.
Full-text available
Since their discovery in 2006, induced pluripotent stem cells (iPSCs) have opened up a world of possibilities for regenerative medicine and novel cell-based therapeutics. Now, over a decade later, robust reprogramming and expansion and differentiation protocols have been developed, and iPSC-derived cells have been used in a wide variety of small and large animal models to treat many different diseases. Furthermore, the first iPSC derivatives are on their way into clinical trials. In this line, (i) GMP-compliant generation, cultivation, and differentiation, (ii) preclinical efficacy and safety, as well as (iii) ethical and regulatory compliance of stem cell research represent important aspects that need to be evaluated for proper clinical translation of iPSCs and their derivatives. In this review article, we provide an overview of the current advances and challenges of the clinical translation of iPSC-derived blood cells and highlight the most pressing problems that have to be overcome in the next years. Stem Cells Translational Medicine 2018. © 2018 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press
Full-text available
The term “Endothelial progenitor cell” (EPC) has been used to describe multiple cell populations that express endothelial surface makers and promote vascularisation. However, the only population that has all the characteristics of a real “EPC” is the Endothelial Colony Forming Cells (ECFC). ECFC possess clonal proliferative potential, display endothelial and not myeloid cell surface markers, and exhibit pronounced postnatal vascularisation ability in vivo. ECFC have been used to investigate endothelial molecular dysfunction in several diseases, as they give access to endothelial cells from patients in a non-invasive way. ECFC also represent a promising tool for revascularization of damaged tissue. Here we review the translational applications of ECFC research. We discuss studies which have used ECFC to investigate molecular endothelial abnormalities in several diseases and review the evidence supporting the use of ECFC for autologous cell therapy, gene therapy and tissue regeneration. Finally, we discuss ways to improve the therapeutic efficacy of ECFC in clinical applications, as well as the challenges that must be overcome to use ECFC in clinical trials for regenerative approaches.
Full-text available
Chronic wounds are a major complication in patients with cardiovascular diseases. Cell therapies have shown potential to stimulate wound healing, but clinical trials using adult stem cells have been tempered by limited numbers of cells and invasive procurement procedures. Induced pluripotent stem cells (IPSCs) have several advantages of other cell types, for example they can be generated in abundance from patients' somatic cells (autologous) or those from a matched donor. IPSCs can be efficiently differentiated to functional endothelial cells (IPSC-ECs). Here, we used a murine excisional wound model to test the pro-angiogenic properties of iPSC-ECs in wound healing. Two full-thickness wounds were made on the dorsum of NOD-SCID mice and splinted. IPSC-ECs (5x105) were topically applied to one wound, with the other serving as a control. Treatment with iPSC-ECs significantly increased wound perfusion and accelerated wound closure. Expression of endothelial cell (EC) surface marker, PECAM-1 (CD31), and pro-angiogenic EC receptor, Tie1, mRNA was upregulated in iPSC-EC treated wounds at 7 days post-wounding. Histological analysis of wound sections showed increased capillary density in iPSC-EC wounds at day 7 and day 14 post-wounding, and increased collagen content at day 14. Anti-GFP fluorescence confirmed presence of iPSC-ECs in the wounds. Bioluminescent imaging showed progressive decline of iPSC-ECs over time, suggesting that iPSC-ECs are acting primarily through short-term paracrine effects. These results highlight the pro-regenerative effects of iPSC-ECs and demonstrate that they are a promising potential therapy for intractable wounds.
Full-text available
Statement of significance: The basic requirements of biomaterial-based nerve guidance conduits have now been well established and include being able to bridge a nerve injury to support macroscopic guidance between nerve stumps, while being strong enough to withstand longitudinal tension and circumferential compression, in addition to being mechanically sound to facilitate surgical handling and implantation. While meeting these criteria, conduits are still limited to the treatment of small defects clinically and might benefit from additional biochemical stimuli to enhance repair for the effective treatment of larger injuries. In this study, a gene activated conduit was successfully developed by incorporating non-viral nanoparticles capable of efficient Schwann cell and neuronal cell transfection with therapeutic genes in vitro, which showed potential to enhance repair in future applications particularly when taking advantage of the transcription factor c-Jun. This innovative approach may provide an alternative to conduits used as platforms for the delivery neurotrophic factors or genetically modified cells (viral gene therapy), and a potential solution for the unmet clinical need to repair large peripheral nerve injury effectively.
Full-text available
Background: Angiogenesis is of utmost importance for tissue regeneration and repair. Human dental pulp stromal cells (hDPSCs) possess angiogenic potential, as they secrete paracrine factors that may alter the host microenvironment. However, more insight into how hDPSCs guide endothelial cells (ECs) in a paracrine fashion is yet to be obtained. Therefore, the current study aimed to investigate the effect(s) of conditioned medium derived from hDPSCs (hDPSC-CM) on EC behavior in vitro. Methods: hDPSCs were harvested from third molars scheduled for surgical removal under informed consent. The angiogenic profile of hDPSC-CM was identified using human angiogenesis antibody array and enzyme-linked immunosorbent assay (ELISA). Using real-time reverse transcription-polymerase chain reaction (RT-PCR) and ELISA, the mRNA and protein expression level of specific angiogenic biomarkers was determined in human umbilical vein endothelial cells (HUVECs) exposed to hDPSC-CM. The effect of hDPSC-CM on HUVEC attachment, proliferation and migration was evaluated by crystal violet staining, MTT, transwell migration along with real-time cell monitoring assays (xCELLigence; ACEA Biosciences, Inc.). A Matrigel assay was included to examine the influence of hDPSC-CM on HUVEC network formation. Endothelial growth medium (EGM-2) and EGM-2 supplemented with hDPSC-CM served as experimental groups, whereas endothelial basal medium (EBM-2) was set as negative control. Results: A wide range of proangiogenic and antiangiogenic factors, including vascular endothelial growth factor, tissue inhibitor of metalloproteinase protein 1, plasminogen activator inhibitor (serpin E1), urokinase plasminogen activator and stromal cell-derived factor 1, was abundantly detected in hDPSC-CM by protein profiling array and ELISA. hDPSC-CM significantly accelerated the adhesion phases, from sedimentation to attachment and spreading, the proliferation rate and migration of HUVECs as shown in both endpoint assays and real-time cell analysis recordings. Furthermore, Matrigel assay demonstrated that hDPSC-CM stimulated tubulogenesis, affecting angiogenic parameters such as the number of nodes, meshes and total tube length. Conclusions: The sustained proangiogenic and promaturation effects of hDPSC-CM shown in this in vitro study strongly suggest that the trophic factors released by hDPSCs are able to trigger pronounced angiogenic responses, even beyond EGM-2 considered as an optimal culture condition for ECs.
Lymphedema is a chronic progressive disease ultimately resulting in severe, disfiguring swelling and permanent changes of the affected tissues. Presently, there is no causal treatment approach of lymphedema. Therefore, most therapies are purely symptomatic. However, the recent use of stem cell-based therapies has offered new prospects for alternative treatment options. The present study was performed to investigate the effects of human adipose-derived stem cells (ADSCs) on human dermal lymphatic endothelial cells (HDLECs) in terms of basic in vitro lymphangiogenic assays (WST-8 assay, scratch assay, transmigration assay, sprouting assay, tube formation assay). The influence of ADSC-conditioned medium (ADSC-CM) on HDLECs was compared to recombinant VEGF-C, bFGF and HGF. Further ADSC-CM was characterized by protein microarray and enzyme-linked immunosorbent assay (ELISA). Although key-lymphangiogenic growth factors - like VEGF-C - could only be detected in low concentrations within the conditioned medium (CM), HDLECs were potently stimulated to proliferate, migrate and to form tube like structures by ADSC-CM. Despite concentrations more than hundredfold higher than those found in the conditioned medium, stimulation with recombinant VEGF-C, bFGF and HGF was still weaker compared to ADSC-CM. These results highlight the effectiveness of growth factors secreted by ADSC to stimulate HDLEC, potentially providing a promising new therapeutic approach for the treatment of lymphedema.
Collagen‐glycosaminoglycan (CG) scaffolds have been widely developed for a range of regenerative medicine applications. To enhance their efficacy, CG scaffolds have previously been pre‐vascularised in vitro using human endothelial cells and mesenchymal stromal cells (hMSC); however, at later time‐points, a regression of vascularisation is observed. This is undesirable for longer pre‐culture periods (e.g., for partial/full organ regeneration) and for in vitro vascularised tissue model systems (e.g., for drug testing/modelling). We hypothesised that delayed platelet‐derived growth factor‐BB (PDGF) addition could stabilise vessels, preventing their regression. In 2D, we identified 25ng/ml as a suitable dose that enhanced hMSC metabolic activity and proliferation, without affecting endothelial cells, or migration in either cell type. In our 3D model of CG scaffold vascularisation, early addition of PDGF (day 3), behaved similarly to no PDGF controls. However, PDGF addition at later time‐points (i.e., day 4 and 5) with a second addition on day 10, prevented vascular regression. In quantifying our observations, we identified a need for a tool to measure in vitro vascularisation in porous scaffolds. This was a second key objective of this work. A novel ImageJ macro was developed, which allowed us to analyse vessel‐like structures, evaluating their number and morphology, and confirmed our qualitative observations. Finally, upregulation of angiogenic genes (ANG1, KDR and TEK2) involved in vessel maturation illustrated how PDGF addition contributed to vascular stability. Taken together, the results suggest that addition of PDGF at specific time‐points can be used to stabilise vasculature in CG scaffolds.
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.