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Biomedicines 2021, 9, 160. https://doi.org/10.3390/biomedicines9020160 www.mdpi.com/journal/biomedicines
Article
SDF-1α Gene-Activated Collagen Scaffold Restores
Pro-Angiogenic Wound Healing Features in Human Diabetic
Adipose-Derived Stem Cells
Ashang L. Laiva
1,2
, Fergal J. O’Brien
1,3,4
and Michael B. Keogh
1,2,
*
1
Tissue Engineering Research Group, Department of Anatomy and Regenerative Medicine, Royal College of
Surgeons in Ireland, 123 St. Stephen’s Green, Dublin 2, Ireland; lluwang@rcsi-mub.com (A.L.L.);
fjobrien@rcsi.ie (F.J.O.)
2
Department of Biomedical Science, Royal College of Surgeons in Ireland, P.O. Box 15503, Adliya, Bahrain
3
Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin,
Dublin 2, Ireland
4
Advanced Materials and Bioengineering Research Centre, Royal College of Surgeons in Ireland and Trinity
College Dublin, Dublin 2, Ireland
* Correspondence: mkeogh@rcsi-mub.com; Tel.: +973-17351450
Abstract: Non-healing diabetic foot ulcers (DFUs) can lead to leg amputation in diabetic patients.
Autologous stem cell therapy holds some potential to solve this problem; however, diabetic stem
cells are relatively dysfunctional and restrictive in their wound healing abilities. This study sought
to explore if a novel collagen–chondroitin sulfate (coll–CS) scaffold, functionalized with polyplex
nanoparticles carrying the gene encoding for stromal-derived factor-1 alpha (SDF-1α
gene-activated scaffold), can enhance the regenerative functionality of human diabetic
adipose-derived stem cells (ADSCs). We assessed the impact of the gene-activated scaffold on
diabetic ADSCs by comparing their response against healthy ADSCs cultured on a gene-free
scaffold over two weeks. Overall, we found that the gene-activated scaffold could restore the
pro-angiogenic regenerative response in the human diabetic ADSCs similar to the healthy ADSCs
on the gene-free scaffold. Gene and protein expression analysis revealed that the gene-activated
scaffold induced the overexpression of SDF-1α in diabetic ADSCs and engaged the receptor
CXCR7, causing downstream β-arrestin signaling, as effectively as the transfected healthy ADSCs.
The transfected diabetic ADSCs also exhibited pro-wound healing features characterized by active
matrix remodeling of the provisional fibronectin matrix and basement membrane protein collagen
IV. The gene-activated scaffold also induced a controlled pro-healing response in the healthy
ADSCs by disabling early developmental factors signaling while promoting the expression of
tissue remodeling components. Conclusively, we show that the SDF-1α gene-activated scaffold can
overcome the deficiencies associated with diabetic ADSCs, paving the way for autologous stem cell
therapies combined with novel biomaterials to treat DFUs.
Keywords: gene-activated scaffold; SDF-1α; human diabetic ADSCs; angiogenesis; wound healing
1. Introduction
Stem cell-driven wound healing is an inherent biological process that occurs to
restore a damaged tissue [1]. The stem cells are recruited in response to signals released
from the wound site such as the stromal-derived factor-1 alpha (SDF-1α) and colony
stimulating factor (CSF) [2,3]. However, in patients with underlying medical conditions
such as diabetes, stem cells recruitment to the wound site is impaired, contributing to
progression of the wound to a more deleterious chronic state [4,5]. In diabetic foot ulcer
patients, poor prognosis poses a serious risk for amputation, quality of life and mortality
Citation: Laiva, A.L.; O’Brien, F.J.;
Keogh, M.B. SDF-1α Gene-Activated
Collagen Scaffold Restores
Pro-Angiogenic Wound Healing
Features in Human Diabetic
Adipose-Derived Stem Cells.
Biomedicines 2021, 9, 160. https://
doi.org/10.3390/biomedicines
9020160
Received: 4 January 2021
Accepted: 18 January 2021
Published: 6 February 2021
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Biomedicines 2021, 9, 160 2 of 24
of the patient [6]. The involvement of many factors such as vascular insufficiency,
neuropathy, susceptibility to infection, local recurrence risk and the propensity to
aggravate presents a major challenge in finding an ultimate treatment of the healing
disorder [4].
Rapid healing is considered the key to prevent amputation in DFU patients [7].
Emerging evidence suggests that the application of tissue-engineered grafts significantly
outperforms the standard of care (i.e., debridement and infection control with regular
dressing changes) in the ability to accelerate healing [8]. Apligraf® and Dermagraft® are
two of the widely used FDA-approved bioengineered constructs for diabetic foot ulcers
[9]. Apligraf® comprises a dermal layer of human neonatal fibroblasts in a bovine type I
collagen matrix and an epidermal layer formed by human neonatal keratinocytes [10],
while Dermagraft® is generated by culturing human neonatal fibroblasts on a
bioresorbable polyglactin scaffold [11]. However, these constructs are not approved for
application over wounds with exposed muscle, tendon or bone [10]. Moreover, the
proportion of patients responding effectively to these grafts is moderate (56% for
Apligraf® [11] and 30% for Dermagraft® [12]. In particular, Apligraf® is also known to
show a relatively short persistence in the wound (~4 weeks), leading to the belief that the
accelerated healing outcome is potentially due to the factors secreted by cells in Apligraf®
[13].
Stem cell therapy is an emerging solution for non-healing wounds. The efficacy of
stem cell therapy is primarily attributed to stem cells’ self-renewal capacity, paracrine
and immunomodulatory effects and the ability to differentiate and remodel the tissue
matrix [14]. Stem cells from allogenic sources including bone marrow [15], adipose tissue
[16] and the umbilical cord [17] have been used to treat DFU patients. One potential
reason for using allogenic stem cells is the impaired functionality of autologous diabetic
stem cells [18–21]. Therapeutic gene delivery to diabetic stem cells is one of the
approaches that might potentially improve the functionality of diabetic stem cells [22].
Conventional gene delivery strategies involve relatively invasive intradermal injections
[23,24]. Genes are often delivered in high amounts (<100 μg) with such strategies. Our
previous studies have found that combining therapeutic genes with biomimetic collagen
scaffolds (gene-activated scaffold) is an effective approach for enhancing tissue
regeneration. The biomimetic scaffold acts a platform for supporting the
three-dimensional growth of the cells while also facilitating their transfection [25,26]. The
use of gene-activated scaffolds can promote a significant healing response with a genetic
cargo dose as low as 2 μg [27]. The enhanced wound healing potency of gene-activated
scaffolds is attributed to their ability to produce controlled spatiotemporal expression of
the therapeutic transgene at the wound site [28].
In our lab, we rely on the use of non-viral-based vectors such as polyethyleneimine
(PEI) for the transfection of cells. PEI possesses high transfection efficiency over a range
of cell types and can easily condense in the presence of plasmid DNA to form highly
stable nanoparticles [28,29]. The process of developing a non-viral-based gene-activated
scaffold first involves formulating nanoparticle complexes of a vector and a plasmid
encoding the therapeutic gene. The nanoparticles are then soak-loaded onto the
biomaterial scaffolds. These nanoparticles sit on the pore’s sidewalls in the scaffold,
where they are taken up by the cells as the cells migrate through the pores [25,26].
Cellular uptake of the nanoparticles leads to functional activation of the cells, which
eventually signals surrounding cells to stimulate local tissue regeneration [25,26].
Common biomimetic scaffolds used for the development of gene-activated scaffolds
include collagen/chondroitin sulfate (coll–CS) [30] and/or hyaluronic acid [31] and
collagen/chitosan [32] that are known to exhibit potent wound healing properties. A
schematic of the development of a gene-activated scaffold is presented in Figure 1A.
Multiple genes (e.g., bone morphogenic protein-2 and VEGF) can also be delivered
through the gene-activated scaffold to further enhance stem cell differentiation and tissue
repair in vivo [27]. Alternatively, the gene-activated scaffold can be functionalized with
Biomedicines 2021, 9, 160 3 of 24
silencing RNAs (e.g., transforming growth factor- beta 1) to limit unwanted healing
outcomes such as scarring in skin regeneration [33].
Figure 1. Gene-activated scaffold for wound healing applications and its impact on human adipose-derived stem cells
(ADSCs). (A) A schematic of the application of a gene-activated scaffold for wound healing. Gene-activated scaffolds can
be directly implanted into the wound where host cells infiltrating the gene-activated scaffold induce the reparative
effects. Alternatively, the tissue engineering approach can be adopted where stem cells are grown on the gene-activated
scaffold and the resulting construct is implanted into the wound site. (B) Relative expression of functional factors
produced by human diabetic ADSCs on the gene-free scaffold compared to healthy ADSCs on the gene-free scaffold on
day 7. An angiogenesis proteome profiler was used to screen the contents of the ADSCs secretome. Conditioned media
(CM) were pooled in equal volumes from the three replicates to assay the proteome. Expression levels were quantified
based on mean volume intensities determined using a ChemiDoc system. The diabetic ADSCs on the gene-free scaffold
produced elevated levels of anti-angiogenic factors (PAI-1, TIMP-1, PEDF and TSP-1), inflammatory cytokine IL-8 and
Biomedicines 2021, 9, 160 4 of 24
vascular disruptive factor Ang-2 relative to their healthy equivalent. (C) Representative blot images of the proteome
analysis.
Stem cells from bone marrow (BM-MSCs) are often the cell candidates while
developing gene-activated scaffold-based therapeutic modalities [34–36]. A recent study
by Kolakshyapati et al. also showed that an epidermal growth factor gene-activated
scaffold enhanced bone marrow stem cells’ differentiation into sweat gland-like cells,
leading to the regeneration of sweat gland-like structures in vivo [36]. However, diabetic
patients suffer from reduced stem cell populations in bone marrow [37], suggesting that
BM-MSCs may not be ideal for developing personalized tissue-engineered products for
diabetic patients [38]. Human umbilical cord blood stem cells are another attractive stem
cell candidate. They have also been approved by the FDA but only for the treatment of
blood-related disorders [39]. An example product is CLEVECORDTM [40]. However, the
stem cells yield in the cord blood is very low and their functionality is often limited by
poor engraftment in the host tissue [39]. In this regard, adipose tissue represents a more
desirable source for harvesting autologous stem cells. The yield capacity of stem cells
from adipose tissue (ADSCs) could be as high as 500 times that of the stem cells derived
from the same mass of bone marrow [41]. Moreover, the stem cells from adipose tissue
could be harvested using a minimally invasive liposuction process [41]. Alofisel® and
Queencell® are examples of ADSCs-based products approved for use in Europe and
South Korea, respectively, for the treatment of Crohn’s disease [40]. Recently,
ADSCs-loaded hydrogel sheets (ALLO-ASC-sheet; Anterogen, South Korea) have also
been shown to enhance healing in DFU patients [42].
Biomaterial scaffolds developed from the copolymer of type I collagen and
chondroitin sulfate (coll–CS) are some of the most clinically efficacious scaffolds used for
wound healing [30]; for example, Integra’s dermal regeneration template, which also
received FDA approval for use in DFU treatment [43]. Our lab uses an optimized
freeze-drying protocol to produce coll–CS scaffolds with highly uniform pore
architectures, which facilitates efficient cell adhesion and infiltration within the scaffold
[44]. Recently, we showed that coll–CS can be functionalized with nanoparticles carrying
the pro-angiogenic gene SDF-1α (SDF-1α gene-activated scaffold) and significantly
enhance the pro-angiogenic responses in BM-MSCs and human Schwann cells [34,45].
SDF-1α primarily functions as a chemokine to recruit endothelial progenitors at the
wound site to promote angiogenesis [46]. However, it is deficient in diabetic wounds [46]
and is known to localize predominantly at the wound margins [47]. Moreover, SDF-1α is
also known to exert a pro-survival effect in ADSCs [48]. Therefore, we sought to
investigate if the SDF-1α gene-activated scaffold could be used to restore the
regenerative potential of human diabetic ADSCs and engineer a functionally enhanced
graft for wound healing. Having initially established that diabetic stem cells are
functionally impaired [18–21], we investigated the functional improvement in diabetic
ADSCs with reference to healthy ADSCs cultured on the gene-free coll–CS scaffold. The
functional improvement was determined based on the production of an array of
angiogenic/anti-angiogenic factors, angiogenic bioactivity of the secreted factors and the
expression patterns of extracellular matrix genes and proteins essential for wound
healing.
2. Materials and Methods
2.1. 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 first amplified by
transforming chemically competent DH5α E. coli cells (Biosciences, Ireland) according to
the manufacturer’s 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, bacterial colonies were harvested and amplified in LB broth
Biomedicines 2021, 9, 160 5 of 24
containing the appropriate antibiotic and cultured overnight in a shaker incubator at 37
°C. Plasmid purification was performed using a QIAGEN® EndoFree®Plasmid Maxi kit
(Qiagen, Sussex, United Kingdom) and final nucleic acid concentration was determined
using NanoDrop 1000 spectroscopy. Plasmids were further diluted in TE buffer to obtain
a working concentration of 0.5 μg/μL and stored at −20 °C until use. Plasmid DNA
(pDNA) encoding a non-therapeutic Gaussia luciferase (pLuc) purchased from New
England Biolabs, Massachusetts, USA, was similarly amplified using ampicillin as the
selective antibiotic. Based on our previous study [34], polyplex particles were formulated
by initially mixing a specified amount of branched cationic 25 kDa PEI (Sigma-Aldrich,
Ireland) and anionic pDNA (fixed at a dose of 2 μg) to give an N/P ratio of 10.
2.2. Cell Expansion
Human ADSCs (healthy, female, age 33, Cat no. 10HU-001, Lot no. 200359; diabetic
type 2, female, age 45, Cat no.10HU-007, Lot no. 200404) purchased from iXCells
Biotechnologies, were expanded to passage 4 in ADSCs growth medium (Cat no.
MD0003) supplied by the company.
2.3. Cell Seeding on SDF-1α Gene-Activated Scaffold
Solid porous coll–CS scaffolds were first developed by freeze-drying a blend
solution of collagen type I and chondroitin sulfate, using the optimized protocol
developed in our lab [49,50]. The scaffolds were then dehydrothermally treated under
vacuum at 105 °C and further crosslinked using
14 mM N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride and
5.5 mM N-Hydroxysuccinimide (EDAC/NHS) (Sigma, UK) to mechanically reinforce the
scaffolds. Using these gene-free coll–CS scaffolds, a preliminary test group was created
by culturing healthy or diabetic ADSCs. Briefly, the scaffolds were hydrated in PBS and
placed in a 12-well plate. The ADSCs at a total density of 5 × 105 cells (2.5 × 105 per side)
were then seeded onto the scaffolds. After letting the cells settle for about 20 min, 2 mL of
OptiMEM (transfection media) was added, and the cellularized scaffolds were incubated
at 37 °C for 24 h. For the final test group, an SDF-1α gene-activated scaffold was
developed, which involved soak-loading the PEI-pSDF-1α polyplex nanoparticles into
the freeze-dried scaffolds. Healthy/diabetic ADSCs at the same cell density as on the
gene-free scaffold were seeded and incubated in OptiMEM. In this experimental run,
healthy ADSCs on the gene-free scaffold were used as control. After incubation in
OptiMEM for 24 h, the cellularized gene-free or gene-activated scaffolds were transferred
into new 12-well plates and fed with 2 mL of ADSCs growth medium. Media change was
then performed every 3–4 days until day 14 by collecting 1 mL of the conditioned media
(CM) and replacing them with new media. All CM were stored at −80 °C until analysis.
2.4. Proteome Profiling of Secreted Factors from Healthy and Diabetic ADSCs on the Gene-Free
Scaffold
In order to first confirm functional impairment in diabetic ADSCs, secreted factors
produced by diabetic ADSCs were compared against those produced by healthy ADSCs,
using an angiogenesis proteome profiler (ARY007, R&D Biosystems, UK). Based on the
technique adopted in previous studies [51,52], equal volumes of CM pooled from each
replicate (n = 3; 500 μL/group) on day 7 were used for the analysis. The amount of
secreted factors was semi-quantitatively determined from the mean volume intensities
obtained using ChemiDoc XRS+ (Biorad). The high-sensitivity mode of ChemiDoc XRS+
was used to detect the bound analytes on the array. Change in the expression of the
protein was then determined relative to that of the healthy ADSCs on the gene-free
scaffold.
Biomedicines 2021, 9, 160 6 of 24
2.5. qRT-PCR Analysis to Determine Functional Gene Expression in ADSCs on SDF-1α
Gene-Activated Scaffold
In order to determine the activation of functional genes, the ADSCs from the
scaffolds or SDF-1α GAS were harvested on the 7th and 14th days for analysis. The cells
were first lysed using Qiazol reagent (Qiagen, UK) to extract the RNA. Chloroform was
then added to separate the cell lysate into protein, DNA and RNA phases. RNA was
extracted using the RNeasy Kit (Qiagen, UK). The RNA quality and quantity were
determined using a Multiskan Go plate reader (Thermo Scientific, UK) with the
absorbance set at 260 nm. Prior to using a reverse transcriptase enzyme (Qiagen, UK) for
cDNA synthesis, genomic DNA was removed by heating the RNA to 42 °C for 2 min
using a genomic DNA wipeout buffer (Qiagen, UK). qRT-PCR was then performed on
cDNA using the following primers: Hs_CXCL12_1_SG, Hs_CXCR4_1_SG,
Hs_ACKR3_1_SG, Hs_ARRB_1_SG, Hs_FN1_1_SG and Hs_COL4A1_1_SG, which
encode for SDF-1α, CXCR4, CXCR7, β-arrestin, fibronectin and collagen IV, respectively.
Fold change in mRNA expression relative to the respective controls at days 7 and 14 was
calculated using the 2-∆∆CT method from averages of three samples for each group.
Human GAPDH (Hs_GAPDH_1_SG) was used as the housekeeping gene.
2.6. Proteome Profiling of Secreted Factors from Healthy and Diabetic ADSCs on SDF-1α
Gene-Activated Scaffold
To understand how the SDF-1α gene-activated scaffold affected the production of
therapeutic factors in diabetic ADSCs, we adopted the similar profiling method
described in Section 2.4. The secretory profile of the diabetic ADSCs was again compared
to that of the healthy ADSCs on the gene-free scaffold and on the gene-activated scaffold.
2.7. Pro-Angiogenic Bioactivity Analyses of Secreted Factors from the ADSCs on SDF-1α
Gene-Activated Scaffold
Next, to determine the angiogenic impact of secreted factors, human umbilical vein
endothelial cells (HUVECs, Cat no. 10HU-012, iXcells Biotechnologies) were exposed to
CM collected from the ADSCs’ culture on day 7, and the subsequent angiogenic response
by HUVECs in terms of network branching and tubule formation on MatrigelTM
(Corning, UK) was assessed. The HUVECs were seeded at a density of 3 × 104 cells/well of
a 48-well plate pre-coated with 120 μL of Matrigel for 30 min at 37 °C. The angiogenic
response was monitored at 4, 8 and 24 h post-exposure to CM. At 8 h, the mean number
of branching points and tubules was counted using the ImageJ software (ImageJ, NIH,
Maryland, USA). All the images were captured at 10× magnification using an IX73
(Olympus, Japan) inverted microscope.
2.8. Immunofluorescent Imaging
Immunofluorescence staining was performed to detect the expression of target
proteins by ADSCs. Scaffolds harvested at days 7 and 14 were used for the study. The
scaffolds were first washed with PBS and fixed in 10% neutral buffered formalin for 20
min. The fixed samples were then processed using the standard protocol for
paraffinization. The blocks were then cut into 8-μm thick slices and collected on charged
slides. The sections were then deparaffinized using xylene followed by rehydration of the
section with decreasing gradients of ethanol. Subsequently, the cells were permeabilized
with 0.2% Tween®20 (Sigma-Aldrich, France) solution in PBS for 30 min (10 min wash x 3)
and blocked using 10% NGS (Normal Goat Serum, Invitrogen, UK)/5% BSA/0.3M
Glycine (prepared in permeabilizing solution) for 1 h. The slides were briefly rinsed in
PBS and then incubated at 4 °C overnight with antibodies against SDF-1α (rabbit mAb,
1:100, ab155090), CXCR7 (mouse mAb, 1:50, MAB42273), fibronectin (rabbit polyAb,
1:100, ab2413) and collagen IV (rabbit polyAb, 1:100, ab6586). All the primary antibodies
were obtained from Abcam UK, except CXCR7 (R&D systems, UK).
Biomedicines 2021, 9, 160 7 of 24
The next day, the slides were rinsed in PBS thrice for 2–3 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, Invitrogen, UK) at 1:800 dilution at room temperature for 1
h in the dark. The rinsing step was performed as before and counterstained for nuclei
using the mounting medium with DAPI (ab104139, Abcam, UK). The slides were then
imaged using a fluorescence microscope (Olympus BX43, Japan) at 40× objective.
Samples incubated with only secondary antibodies were used as controls. All the
antibodies were diluted in 1% BSA in PBS.
2.9. Image Analysis
The “ImageJ” software (ImageJ, NIH, Maryland, USA) was used to
semi-quantitatively determine the amount of expressed proteins. For each marker, a
constant threshold value was first determined through preliminary imaging of various
sections. Using the set threshold value, integrated density (stained area x mean gray
value) of the images was determined and then normalized to the number of cells (nuclei
counting) to give a final mean fluorescence density per cell. An average was quantified
from 8–12 random non-overlapping images per replicate, with a minimum of 3 replicates
per group. The averages obtained from the 3 replicates/group were then used for
measuring relative expression between the groups.
2.10. Statistical Analysis
All results are expressed as mean ± standard deviation. An unpaired two-tailed t-test
was used to demonstrate the statistical significance between groups, where p < 0.05 was
considered to be significant.
3. Results
3.1. Diabetes Impairs Signaling of Functional Factors in Human ADSCs
Figure 1B shows the relative expression of the functional factors secreted by the
ADSCs on day 7. Overall, the diabetic ADSCs showed elevated production of
inflammatory cytokine IL-8 (2-fold), anti-angiogenic factors PAI-1 (1.8-fold), TIMP-1
(2.9-fold), PEDF (2.2-fold) and TSP-1 (2.2-fold) and vascular destabilizing factor Ang-2
(1.8-fold) relative to their healthy equivalent. Relatively, the diabetic ADSCs also showed
reduced production of MCP-1 by 40%. Meanwhile, the levels of pro-angiogenic factors
(VEGF, ANG and Ang-1) between the two groups were comparable. Therefore, taken
together, this finding verifies that functional signaling is impaired in diabetic ADSCs.
Table 1 provides the functional role of the secreted factors in wound healing.
Table 1. ADSCs’ secreted factors and their role in angiogenic wound healing.
Soluble
Factor Full Name Function
IL-8 Interleukin-8 Recruitment of neutrophils; component of early
inflammatory phase; angiogenic [53,54]
MCP-1
Monocyte
chemoattractant
protein-1
Recruitment of monocytes; essential for progression of
healing; angiogenic [53,55]
PAI-1 Plasminogen activator
inhibitor-1
Serine protease inhibitor; inhibits fibrinolysis; promotes
re-epithelialization; anti-angiogenic [56,57]
uPA Urokinase-type
plasminogen activator Mediates fibrinolysis; promotes angiogenesis [58,59]
VEGF Vascular endothelial
growth factor Pro-angiogenic factor; stimulates vessel sprouting [60]
Biomedicines 2021, 9, 160 8 of 24
ANG Angiogenin Exerts ribonuclease activity; regulates VEGF-induced
endothelial proliferation and angiogenesis [61]
Ang-2 Angiopoietin-2 Promotes vessel sprouting with VEGF; exerts vascular
destabilizing effects [62,63]
Ang-1 Angiopoietin-1 Exerts vascular protective effects; essential for vessel
stabilization [64]
TIMP-1 Tissue inhibitor of
metalloproteinase-1
Anti-angiogenic; involved in the regeneration of
epidermis and matrix remodeling [65–67]
PEDF
Pigment
epithelium-derived
factor
Potent anti-angiogenic factor; expression increases at
late stage of wound healing; promotes resolution of
angiogenesis [68]
TSP-1 Thrombospondin-1 Matricellular protein; inhibits angiogenesis; elevated
expression suppresses wound healing [69,70]
3.2. SDF-1α Gene-Activated Scaffold Promotes Overexpression of SDF-1α mRNA and Engages
the CXCR7/β-Arrestin Signaling in Diabetic ADSCs
Gene expression analysis first showed that the SDF-1α gene-activated scaffold
caused the overexpression of SDF-1α mRNA in diabetic ADSCs, similar to the transfected
healthy ADSCs. Having observed this, we then looked for the receptors associated with
SDF-1α signaling. Of the two primary receptors for SDF-1α—CXCR4 and CXCR7—we
could only detect the expression of CXCR7 mRNA (Figure 2). The healthy ADSCs on the
gene-activated scaffold demonstrated significantly (p < 0.01) higher (63%) expression of
CXCR7 mRNA than their gene-free scaffold equivalent. However, the diabetic ADSCs,
despite overexpressing SDF-1α mRNA, showed a significantly (p < 0.005) lower (70%)
activation of CXCR7 mRNA than the healthy ADSCs on the gene-free scaffold.
Immunofluorescence imaging then showed that all the ADSCs abundantly
expressed SDF-1α proteins; however, contrary to the gene expression data, no obvious
differences in the expression were observed. The diabetic ADSCs on the gene-activated
scaffold showed the highest SDF-1α expression, while the expression of CXCR7 was
comparable to that of the healthy ADSCs on the gene-free scaffold. The transfected
healthy ADSCs, on the contrary, showed a significantly lower (p < 0.001; 61%) expression
of CXCR7 than their gene-free scaffold equivalent.
Next, we assessed the activation of β-arrestin, which is the major downstream signal
transducer of the SDF-1α/CXCR7 axis [71]. We found that the overexpression of SDF-1α
in both healthy and diabetic ADSCs significantly enhanced the expression of β-arrestin
mRNA (1.91 ± 0.33 for healthy; 1.88 ± 0.5 for diabetic) (p < 0.05) compared to that of the
healthy ADSCs on the gene-free scaffold.
Biomedicines 2021, 9, 160 9 of 24
Figure 2. Impact of SDF-1α gene-activated scaffold on the activation of SDF-1α and its downstream signaling mediators
in diabetic ADSCs. (A) SDF-1α gene-activated scaffold caused the overexpression of SDF-1α mRNA in both healthy and
diabetic ADSCs. The overexpression of SDF-1α mRNA had a minimal effect on the expression of CXCR7 mRNA in
diabetic ADSCs, while it increased significantly in healthy ADSCs. (B) Immunofluorescence images showing the
abundancy of SDF-1α and CXCR7 in the ADSCs groups. (C) The diabetic ADSCs on the SDF-1α gene-activated scaffold
expressed the highest level of SDF-1α and CXCR7 proteins, while healthy ADSCs displayed the weakest expression of
CXCR7. *, **, *** and **** indicate statistical significance at p < 0.05, p < 0.01, p < 0.005 and p < 0.001, respectively. Data are
presented as mean ± standard deviation (n = 3). Scale bar 20 μm.
Biomedicines 2021, 9, 160 10 of 24
3.3. SDF-1α Gene-Activated Scaffold Restores a Healthy-Like Signaling of Functional Factors in
Diabetic ADSCs
Having determined that the SDF-1α gene-activated scaffold could effectively engage
the SDF-1α/CXCR7 axis in the diabetic ADSCs, we then assessed if the SDF-1α signaling
enhances the diabetic ADSCs’ functionality. Subsequently, we used proteome profiling to
determine the functional activation of diabetic ADSCs. We noted that the diabetic ADSCs
on the gene-activated scaffold showed markedly improved signaling of the functional
factors, whose pattern closely resembled that of the healthy ADSCs on the gene-free
scaffold (Figure 3A). However, the transfected diabetic ADSCs did not downregulate the
production of its inflammatory cytokine IL-8. The activated diabetic ADSCs instead
showed enhanced production of MCP-1 to a level comparable to IL-8 (Figure 3B).
On the other hand, the healthy ADSCs on the gene-activated scaffold showed a
markedly reduced production of uPA, MCP-1 and VEGF. The transfected healthy ADSCs
also showed a moderate shift in the pattern of production of anti-angiogenic factors. For
instance, the production of TIMP-1 increased by 51%, while the production of TSP-1
decreased by 31% relative to that produced by their gene-free scaffold equivalent.
Biomedicines 2021, 9, 160 11 of 24
Figure 3. Impact of SDF-1α gene-activated scaffold on the production of functional factors in healthy and diabetic ADSCs.
(A) Transfection of the diabetic ADSCs within the SDF-1α gene-activated scaffold resulted in restoration of a healthy-like
signaling of functional factors in the diabetic ADSCs. On the other hand, the SDF-1α gene-activated scaffold caused a
moderate deviation in the signaling pattern of the functional factors in the healthy ADSCs relative to its unactivated
equivalent. (B) Representative blot images of the proteome analysis.
3.4. Diabetic ADSCs on the SDF-1α Gene-Activated Scaffold Effectively Enhance Angiogenesis in
Endothelial Cells
One of the significant challenges in the healing of diabetic wounds is the lack of
angiogenesis [72]. Therefore, having noted an enhanced pro-angiogenic profile in
Biomedicines 2021, 9, 160 12 of 24
transfected diabetic ADSCs, we assessed the pro-angiogenic bioactivity of the secreted
factors on endothelial cells. Treatment of the endothelial cells with CM from the
transfected diabetic ADSCs promoted the formation of well-defined endothelial tubular
networks by 8 h post-treatment (Figure 4A ii). The mean number of branching points and
tubules was 43 ± 8 and 62 ± 8, respectively. The angiogenic response was relatively milder
(39 ± 9 branching points; 56 ± 6 tubules) when stimulated with CM from healthy ADSCs
on the gene-free scaffold. The angiogenic response was further lower (20 ± 3 branching
points; 32 ± 9 tubules) in the endothelial groups exposed to CM from the transfected
healthy ADSCs. Comparatively, the diabetic ADSCs on the SDF-1α gene-activated
scaffold demonstrated significantly (p < 0.05) superior pro-angiogenic potency than their
healthy counterpart.
Figure 4. Pro-angiogenic impact of secreted factors from the transfected healthy and diabetic ADSCs. (A) The diabetic
ADSCs on the SDF-1α gene-activated scaffold induced the strongest pro-angiogenic response in human endothelial
cells. (B) At 8 h post-exposure, CM from diabetic ADSCs significantly enhanced endothelial network branching (p < 0.01)
as well as tubules formation (p < 0.05), compared to that induced by their healthy counterpart. EndoGro Media (EGM) +
VEGF was used as reference medium to stimulate endothelial angiogenesis. Scale bar 100 μm.
Biomedicines 2021, 9, 160 13 of 24
3.5. SDF-1α Gene-Activated Scaffold Promotes Pro-Wound Healing Matrix Remodeling Response
in Diabetic ADSCs
Fibronectin is one of the first matrix proteins produced during the early stages of
cellular development. It also acts as a provisional scaffold for subsequent matrix
deposition [73]. Therefore, we first assessed the expression of the fibronectin gene and
deposition of its matrix. On day 7, the diabetic ADSCs on the gene-activated scaffold
showed a significantly enhanced transcription of the FN1 gene than the healthy ADSCs
on the gene-free scaffold (Figure 5). Immunofluorescence analysis of matrix deposition
further showed that the diabetic ADSCs abundantly deposited the matrix similar to that
observed in the healthy ADSCs on the gene-free scaffold (Figure 6). However, on day 14,
the fibronectin matrix remodeled into thin, continuous fibers, causing an overall
reduction in the spatial coverage of 40% compared that of the healthy ADSCs on the
gene-free scaffold. Conversely, the healthy ADSCs on the gene-activated scaffold did not
show activation of the FN1 gene on day 7 but moderately downregulated its expression
by 45% compared to that of their gene-free scaffold equivalent on day 14. At both time
points, fibronectin deposition by the healthy ADSCs on the gene-activated scaffold was
significantly (p < 0.05) lower than that on the gene-free scaffold equivalent (Figure 6B).
We next assessed the expression of collagen IV, which is essential for the formation
of the basement membrane [74], a specialized structure that binds the dermis to the
epidermis. Conversely to FN1 gene expression, we noted a significant (p < 0.01) early
activation (day 7) of the COL4A1 gene only in the healthy ADSCs on the gene-activated
scaffold. Furthermore, matrix deposition also increased significantly in the healthy
ADSCs on the gene-activated scaffold compared to that on the gene-free scaffold
equivalent. Meanwhile, the diabetic ADSCs on the gene-activated scaffold showed a
temporal increase in the expression of the COL4A1 gene. On day 14, the expression of the
COL4A1 gene by the diabetic ADSCs significantly (p < 0.005) exceeded that of the healthy
ADSCs on the gene-free scaffold. However, at the protein level, the diabetic ADSCs on
the gene-activated scaffold deposited comparable amounts of the collagen IV matrix to
those of the healthy ADSCs on the gene-free scaffold.
Taking the results together, we show that the SDF-1α gene-activated scaffold
restores the pro-regenerative capacity in diabetic ADSCs similar to the healthy ADSCs on
the gene-free scaffold. Further, we show that the SDF-1α gene-activated scaffold also
promotes controlled development of healthy ADSCs towards a pro-healing nature.
Figure 7 depicts the overall findings of this study.
Biomedicines 2021, 9, 160 14 of 24
Figure 5. Effect of SDF-1α gene-activated scaffold on the expression of pro-wound healing matrix genes in healthy and
diabetic ADSCs. (A) On day 7, the transfected diabetic ADSCs on the gene-activated scaffold showed a significantly (p <
0.05) enhanced transcription of the FN1 gene than the healthy ADSCs on the gene-free scaffold. (B) At the same time
point, the healthy ADSCs on the gene-activated scaffold showed a significant activation of the COL4A1 gene than their
gene-free equivalent. *, ** and *** indicate statistical significance at p < 0.05, p < 0.01 and p < 0.005, respectively. Data are
presented as mean ± standard deviation (n = 3).
Biomedicines 2021, 9, 160 15 of 24
Figure 6. Effect of SDF-1α gene-activated scaffold on the deposition and remodeling of pro-wound healing matrix
proteins in healthy and diabetic ADSCs. (A) Relative to the healthy ADSCs on the gene-free scaffold, diabetic ADSCs on
the SDF-1α gene-activated scaffold showed a significant decrease in the deposition of fibronectin matrix while increasing
the deposition of collagen IV over time. Contrarily, healthy ADSCs on the SDF-1α gene-activated scaffold deposited
minimal amounts of the fibronectin matrix throughout the culture period but significantly increased the deposition of
collagen IV. (B) Semi-quantitative interpretation of spatiotemporal expression of the matrix proteins. * and ** indicate
statistical significance at p < 0.05 and p < 0.01, respectively. Data are presented as mean ± standard deviation (n = 3). Scale
bar 20 μm.
Biomedicines 2021, 9, 160 16 of 24
Figure 7. A schematic of the functional changes induced by the SDF-1α gene-activated collagen scaffold in healthy and
diabetic ADSCs. The diabetic ADSCs on the gene-free scaffold initially showed an impaired functional response
characterized by elevated inflammatory cytokine production (IL-8), anti-angiogenic factors (PAI-1, TIMP-1, PEDF and
TSP-1) and vascular destabilizing factor Ang-2 compared to the healthy ADSCs on the gene-free scaffold. However, when
activated within the gene-activated scaffold, the impaired signaling in the diabetic ADSCs could be restored to a
healthy-like state and exert therapeutic paracrine effects capable of enhancing angiogenesis. Meanwhile, the
gene-activated scaffold drove the healthy ADSCs towards an advanced cellular maturation stage while facilitating the
bypass of early signaling events such as the production of uPA, VEGF and MCP-1 and the deposition of the provisional
matrix fibronectin.
4. Discussion
In this study, we explored the therapeutic impact of an SDF-1α gene-activated
collagen scaffold on human diabetic ADSCs as an approach to develop a functional
three-dimensional autologous graft for diabetic wound healing. We found that the
SDF-1α gene-activated scaffold restored pro-angiogenic signaling in the diabetic ADSCs
similar to the healthy ADSCs on the gene-free scaffold. The transfected diabetic ADSCs
also exhibited active matrix remodeling events characterized by a reduction in the
deposition of the fibronectin matrix and an increase in the expression of basement
membrane protein collagen IV. Meanwhile, in healthy ADSCs, the SDF-1α gene-activated
scaffold promoted controlled cellular maturation, by instructing the ADSCs to disable the
signaling of early developmental factors and promote the production of tissue
remodeling components crucial for wound healing.
The impaired healing response in diabetic patients is attributed to the reduced
regenerative potential of the endogenous cells [46]. Diabetic ADSCs often show reduced
expression of pro-angiogenic factors, and this is considered a significant factor limiting
their application for cellular therapy [18,21,75,76]. Nevertheless, wound healing is a
complex process controlled by a cocktail of signaling factors including anti-angiogenic
factors [77]. Therefore, we used an angiogenesis proteome profiler that enabled us to
differentiate between the multiple signaling factors produced by the healthy and diabetic
ADSCs when grown in a three-dimensional scaffold. We noted that it is not necessarily
the lack of a pro-angiogenic component but an impaired production of anti-angiogenic
and vascular destabilizing factors that may also limit the functional potency of ADSCs
(Figure 1). Other studies have also found the association of elevated levels of
anti-angiogenic factors such as PAI-1 [78] and PEDF [79] with poor healing response in
Biomedicines 2021, 9, 160 17 of 24
diabetic wounds. An imbalanced ratio of Ang-2 with Ang-1 has also been implicated as a
predictor of poor healing outcomes in diabetic wounds [80,81].
Subsequently, we sought to assess if the SDF-1α gene-activated scaffold could
restore the diabetic ADSCs’ impairment to an improved functional state. As anticipated,
we noted that SDF-1α gene delivery using the gene-activated scaffold restores the
impairment in secretome production in the diabetic ADSCs as in healthy ADSCs on the
gene-free scaffold (Figure 3). Stem cells’ secretome is a key component for driving wound
healing by instructing surrounding cells such as the endothelial cells and modulating the
wound environment [82]. Therefore, the resulting normalization of the diabetic ADSCs
induced by the gene-activated scaffold may offer enhanced healing capabilities in
autologous treatment strategies. We also proved, in vitro, that the secreted factors from
the diabetic ADSCs on the gene-activated scaffold can stimulate angiogenic growth in
endothelial cells at a similar capacity as that of the healthy ADSCs on the gene-free
scaffold (Figure 4). Moreover, in tissue engineering strategies, a construct’s ability to
induce an enhanced angiogenic response is crucial for faster integration of the graft with
the host environment [83].
Among the secreted factors, a notable response induced by the gene-activated
scaffold in the diabetic ADSCs is the elevated production of MCP-1. In diabetic wounds,
the lack of MCP-1 is considered a major factor halting the progression of healing [84,85].
Wood et al. showed that early treatment with MCP-1 at the time of injury significantly
enhanced macrophage infiltration, thereby accelerating healing in diabetic mice [84].
Investigations on healthy wounds also found that the elevation of MCP-1 sequential to
IL-8 is crucial to drive an acute-like healing [53]. Therefore, despite the production of
high levels of IL-8, the ability of the transfected diabetic ADSCs to equilibrate the level of
MCP-1 to IL-8 may facilitate a timely transition to the subsequent healing phase.
While the SDF-1α gene-activated scaffold works to improve angiogenic homeostasis
in diabetic ADSCs, it appears to disable the signaling components of the early healing
phase in healthy ADSCs. For instance, in healthy ADSCs, the SDF-1α gene-activated
scaffold robustly dampened the production of pro-inflammatory MCP-1 and
pro-angiogenic VEGF. However, it did not compromise the signaling of the
anti-angiogenic tissue inhibitors, which are essential for tissue remodeling [57,67,68,70].
It has been observed that stem cells possess a unique ability to sense external stimuli
and accordingly modulate their secretome to offer a cytoprotective effect [86]. In our
study, the modulatory impact of the SDF-1α gene-activated scaffold in the ADSCs
appears to be controlled by the receptor CXCR7 (Figure 2C). A recent study that
investigated the impact of SDF-1α on the differentiation potential of embryonic stem
cells (ESCs) showed that the presence of active or inactive CXCR7 differentially
modulates the development of ESCs [87]. The group showed that the engagement of
active CXCR7 by SDF-1α leads to downregulation of pluripotency markers without
affecting the expression of factors essential for development in wild-type ESCs [87].
Conversely, in mutant cells with inactive CXCR7, the expression of the pluripotency
markers was barely affected [87]. Similarly, given that diabetic ADSCs are relatively
dysfunctional, the sensitivity of CXCR7 to SDF-1α is probably weaker, leading to a
varied response than transfected healthy ADSCs. Nevertheless, activation of CXCR7 is
known to promote survival and proliferation of ADSCs [17].
Since CXCR4 is the classical receptor of SDF-1α [17,88], we anticipated that the
overexpression of SDF-1α induced by the gene-activated scaffold would upregulate the
expression of CXCR4 in the transfected ADSCs. However, we barely detected the
expression of CXCR4 mRNAs even in the untransfected healthy ADSCs. A similar case
has been observed in MSCs [89], but we cannot rule out that the gene is completely
degraded, as it would lead to cell death [90]. It has been observed that the CXCR4 gene is
generally expressed at very low levels in human ADSCs and is also the least expressed
among the set of chemokine receptor genes [91]. Another factor for the undetection of
CXCR4 could be the activation of CXCR7. The activation of CXCR7 can dampen the
Biomedicines 2021, 9, 160 18 of 24
expression of CXCR4 [92]. However, the mechanism that triggered the activation of
CXCR7 over CXCR4 even in the untransfected ADSCs in the gene-free scaffold needs
further investigation. Nevertheless, a study by Lisignoli et al. [93] suggests that the
matrix may modulate the expression of chemokine receptors. They found that the
transcriptional expression of SDF-1α and CXCR4 is reversed when the MSCs are cultured
on a hyaluronic acid-based scaffold compared to a plastic substrate [93]. The MSCs on the
hyaluronic acid scaffold showed downregulation of SDF-1α mRNA while upregulating
CXCR4 mRNA relative to the MSCs on the plastic substrate [93]. The group found that
the activation of CD54, a cell surface receptor of hyaluronan, on the MSCs modulated this
response [93]. In our context, the ADSCs are known to express the collagen receptor α2β1
integrin [94]. The receptor α2β1 integrin promotes the adhesion and proliferation of stem
cells on collagen [95]. Additionally, CXCR7 is essential for the promotion of proliferation
and survivability in ADSCs [17,96]. Therefore, assuming the regulatory role of the matrix,
it appears that a proliferative mechanism may have been activated upon initial adhesion,
leading to the upregulation of CXCR7 but not CXCR4.
The adipose ECM is also known to possess pro-healing properties [97]. Therefore,
we investigated how the SDF-1α gene-activated scaffold affects diabetic ADSCs in terms
of matrix deposition and remodeling. We focused on the expression and deposition
pattern of two of the traditional matrix proteins—the provisional matrix protein,
fibronectin, and a relatively mature basement membrane protein, collagen IV.
Fibronectin is not only essential for supporting the adhesion and migration of cells but
also provides a scaffold for subsequent collagen deposition [73]. However, diabetic skin
lacks this feature, and this is one of the causes that impede healing [98,99]. To improve
healing, soluble forms of fibronectin are supplemented to the wound to facilitate the
assembly of the fibronectin matrix and promote healing [100,101]. Here, we show that the
SDF-1α gene-activated scaffold effectively enhances the deposition of the fibronectin
matrix by the diabetic ADSCs, imparting the potential to drive healing. Furthermore, the
response that the transfected diabetic ADSCs are driven towards healing could also be
evident from their high transcriptional activity of the COL4A1 gene and assembly of the
collagen IV matrix over time. The SDF-1α gene-activated scaffold also enhanced the
deposition of collagen IV in the healthy ADSCs, suggesting that it promotes cellular
maturation. However, the increase in collagen IV deposition by the transfected healthy
ADSCs occurred despite bypassing the activation of either the FN1 gene or its assembly
as a matrix. Collectively, these events imply that the SDF-1α gene-activated scaffold
promotes controlled remodeling of ADSCs’ ECM to create a pro-healing environment
regardless of the physiological state of the ADSCs. This controlled response in the ADSCs
could be attributed to the homeostatic role of SDF-1α [102].
Specifically, this study addressed a potential solution for developing an enhanced
autologous bioengineered graft using diabetic ADSCs, which are generally impaired.
Patients requiring stem cell therapy could avoid the search for matching healthy donors
and may use their own cells for the treatment. It may both reduce the time of treatment
and costs [103]. However, this study’s limitation is that we did not investigate the
activated diabetic ADSCs’ immunomodulatory effect on immune cells. ADSCs are
known to exert immunosuppressive effects in an inflammatory environment [104].
Therefore, direct or indirect co-culture of immune cells with the activated diabetic ADSCs
will help better understand the therapeutic implications of the gene-activated
scaffold/ADSCs construct. Further studies also need to be performed to determine the
product’s shelf life and potential adverse reactions in vivo, the details of which are crucial
for successful implementation in a hospital setting [105]. Although not approved by the
FDA yet, the gene-activated scaffold components, i.e., the vector PEI [106] and the
SDF-1α plasmid [107], have been safely used in clinical trials. Therefore, we anticipate
that further functional studies using in vivo models will help in more quickly translating
the technology to clinics.
Biomedicines 2021, 9, 160 19 of 24
5. Conclusions
In this study, we report that a three-dimensional collagen–chondroitin sulfate
scaffold functionalized with a pro-angiogenic SDF-1α gene could be used to enhance the
functionality of human diabetic ADSCs as effectively as healthy ADSCs on a gene-free
scaffold. Specifically, overexpression of SDF-1α in the diabetic ADSCs led to
normalization of the production of therapeutic factors, restoring their pro-angiogenic
potency. The diabetic ADSCs also exhibited a pro-healing feature characterized by
active matrix remodeling of fibronectin and collagen IV matrixes. We also note that it
is not necessarily the lack of a pro-angiogenic component but an impaired production of
anti-angiogenic and vascular destabilizing factors that may also limit the functional
potency of diabetic ADSCs. Conclusively, we have shown that a pro-angiogenic collagen
biomaterial can enhance the wound healing response of typically dysfunctional diabetic
ADSCs, paving the way for better patient-specific DFU treatment.
Author Contributions: All planned the experiment, performed the experiments,
analyzed/interpreted the data and wrote the manuscript. FOB and MBK reviewed the data and the
manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This work was funded by RCSI Dilmun PhD scholarship.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable
Data Availability Statement: The datasets used and/or analyzed during the current study are
available from the corresponding author on reasonable request.
Conflicts of Interest: The authors declare that they have no conflict of interests.
List of Abbreviations
ADSCs Adipose-derived stem cells
Ang-2 Angiopoeitin-2
BM-MSCs Bone marrow-mesenchymal stem cells
CSF Colony stimulating factor
Coll–CS Collagen–chondroitin sulfate
CM Conditioned media
ECM Extracellular matrix
EDAC/NHS N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide
hydrochloride/N-Hydroxysuccinimide
FDA Food and Drug Administration
GAS Gene-activated scaffold
HUVECs Human umbilical vein endothelial cells
IGFBP-3 Insulin growth factor binding protein-3
IL-8 Interleukin-8
MCP-1 Monocyte chemoattractant protein-1
PAI-1 Plasminogen activator inhibitor-1
pDNA Plasmid DNA
PEI Polyethyleneimine
PEDF Pigment epithelium-derived factor
SDF-1α Stromal-derived factor-1 alpha
TIMP-1 Tissue inhibitor of metalloproteinase-1
TSP-1 Thrombospondin-1
VEGF Vascular endothelial growth factor
Biomedicines 2021, 9, 160 20 of 24
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