Available via license: CC BY 4.0
Content may be subject to copyright.
pharmaceuticals
Article
Anti-Aging β-Klotho Gene-Activated Scaffold Promotes
Rejuvenative Wound Healing Response in Human
Adipose-Derived Stem Cells
Ashang L. Laiva 1,2 , Fergal J. O’Brien 2,3,4 and Michael B. Keogh 1, 2, *
Citation: Laiva, A.L.; O’Brien, F.J.;
Keogh, M.B. Anti-Aging β-Klotho
Gene-Activated Scaffold Promotes
Rejuvenative Wound Healing
Response in Human
Adipose-Derived Stem Cells.
Pharmaceuticals 2021,14, 1168.
https://doi.org/10.3390/ph14111168
Academic Editors: Takehisa Hanawa,
Yayoi Kawano, Viorica Patrulea,
Mitsutoshi Sato and Olivier Jordan
Received: 29 September 2021
Accepted: 9 November 2021
Published: 17 November 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Tissue Engineering Research Group-Bahrain, Royal College of Surgeons in Ireland, Adliya,
Manama P.O. Box 15503, Bahrain; lluwang@rcsi-mub.com
2Tissue Engineering Research Group, Department of Anatomy and Regenerative Medicine, Royal College of
Surgeons in Ireland, 123 St. Stephen’s Green, D02 YN77 Dublin, Ireland; fjobrien@rcsi.ie
3Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin,
D02 PN40 Dublin, Ireland
4Advanced Materials and Bioengineering Research Centre, Royal College of Surgeons in Ireland and Trinity
College Dublin, D02 YN77 Dublin, Ireland
*Correspondence: mkeogh@rcsi-mub.com
Abstract:
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-
β
3 and TGF-
β
1); (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-
β
3. 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.
Keywords:
anti-aging;
β
-Klotho; gene-activated scaffold; adipose-derived stem cells; angiogenesis;
matrix deposition; rejuvenative healing
1. Introduction
Wound healing is a complex biological process that requires a tight orchestration
of multiple cellular events [
1
]. However, in the aging population, cellular events are
disrupted, leading to delayed healing [
2
]. The disruption in the healing mechanisms arises
partly due to the reduced homing of the progenitor cells from the bone marrow and other
prevailing metabolic diseases such as diabetes [
3
,
4
]. In wound management, the application
of biomaterial scaffolds is becoming more common as a treatment. Biomaterial scaffolds
Pharmaceuticals 2021,14, 1168. https://doi.org/10.3390/ph14111168 https://www.mdpi.com/journal/pharmaceuticals
Pharmaceuticals 2021,14, 1168 2 of 16
protect the wound from infection, absorb wound exudates, and keep the wound moist
to prevent tissue necrosis [
5
]. Therapeutics can also be loaded into biomaterial scaffolds
and promote faster healing in hard-to-heal wounds [
1
]. However, biomaterial scaffolds
alone may not effectively orchestrate the multiple signaling cascades occurring within the
wound. Delivering stem cells is sometimes proposed as a solution for moderating the
complex signaling events in the wound [6].
Stem cells secrete paracrine factors and can differentiate into cells of multiple tissue
lineages [
7
]. Stem cells are typically delivered into the wound through intradermal in-
jections [
6
], topical spraying [
8
], or as tissue-engineered grafts [
9
]. In recent years, the
application of tissue-engineered grafts for chronic wound healing has gradually gained
wider acceptance because of their ability to heal wounds rapidly [
9
–
11
]. Apligraf
®
and
Dermagraft
®
are two of the widely used Food and Drug Administration (FDA)-approved
bioengineered constructs for chronic wound treatment [
12
,
13
]. However, the generation of
these grafts requires prolonged cell culture to produce high cell numbers [
14
]. When using
stem cells, prolonged culture can increase cellular senescence and diminish the stemness of
the stem cells [
15
,
16
]. Therefore, maintaining the stemness of the stem cells is central to
achieving the optimal therapeutic response.
Therapeutic gene delivery to the stem cells using non-viral vectors is a potential
strategy to enhance stem cells’ functionality [
17
]. Traditionally, the cells are transfected
in 2 D cultures and later transplanted
in vivo
[
18
]. However, platforms such as the gene-
activated scaffolds, consisting of biomaterial scaffolds functionalized with nanoparticles
carrying the therapeutic transgene [
19
], that our group have been pioneering offer an
alternative solution for transfecting the cells within a 3 D environment. This study fo-
cused on developing a gene-activated version of the 3 D collagen-chondroitin sulfate
(coll-CS) scaffold that has tremendous clinical translation potential, as its composition
is similar to that of the Integra’s Dermal Regeneration Template (DRT), a clinically ap-
proved scaffold for chronic wound healing [
20
]. We primarily use a cationic polymer called
polyethyleneimine (PEI) to condense DNA plasmids encoding for growth factor genes
such as the stromal-derived factor-1 alpha and nerve growth factor and assemble them into
charged
nanoparticles [21–23].
These charged nanoparticles are then soak-loaded into our
coll-CS scaffold to generate the gene-activated scaffolds. Our previous studies have also
shown that the PEI-based gene-activated scaffold can cause transient overexpression of the
therapeutic transgene in a range of wound-healing cells and promote their regenerative
abilities [
21
,
24
,
25
]. Traditionally, the gene-activated scaffold was developed to target host
cells and promote local repair [
26
]. However, emerging evidence indicates that cell-laden
gene-activated scaffolds bear superior potency to regenerate complex tissue structures than
gene-activated scaffolds alone [27].
Cellular abundance is one of the crucial requirements for the generation of tissue-
engineered grafts [
28
]. The adipose tissue is a rich source that can supply a large number of
stem cells. The adipose tissue contains as many as 500-times the stem cells for the same mass of
bone marrow, a traditional tissue source for stem cell extraction [
29
]. Moreover, the stem cells
can be extracted from the adipose tissue using a minimally invasive liposuction process [
29
].
Our recent study found that the human adipose-derived stem cells (ADSCs) demonstrate
excellent biocompatibility with the coll-CS scaffold. Using a pro-angiogenic gene-activated
scaffold further improved the ADSCs’ regenerative responses [
30
]. However, the ADSCs
tend to lose their stemness upon expansion [
31
]. Previously, we found that an anti-aging
protein
β
-Klotho could significantly improve the proliferation of healthy and diabetic human
ADSCs [
32
]. Most studies generally use the primary variant
α
-Klotho [
33
–
35
] and have shown
that the protein can increase the lifespan of ADSCs by activating the telomerase transcriptional
activity [
33
].
α
-Klotho overexpressing MSCs have also been found to exert strong anti-fibrotic
effects in kidney injury through the inhibition of Wnt/
β
-catenin signaling [
36
]. However, the
role of β-Klotho in stem cells remains relatively unexplored.
Besides the difference in the research focus between the
α
- and
β
-Klotho, the anti-aging
Klotho proteins have not yet been fully incorporated into the tissue engineering strategies
Pharmaceuticals 2021,14, 1168 3 of 16
for wound healing. Therefore, in this study, we sought to study the functional impact of a
β
-Klotho gene-activated scaffold on human ADSCs for wound healing applications. We
first investigated the transcriptional activities of the classical pluripotency factors (Oct-4,
Nanog and Sox-2), proliferation marker (Ki-67) and wound healing regulators (TGF-
β
3
and TGF-
β
1) in the ADSCs. We then assessed the bioactivity of the conditioned media
generated by the ADSCs-laden gene-activated scaffold to evaluate its paracrine potential.
Ultimately, we examined the regeneration of basement membrane (fibronectin, laminin,
and collagen IV) and scar-associated proteins (
α
-SMA and elastin) in the ADSCs-laden
gene-activated scaffold to evaluate controlled tissue maturation.
2. Results
2.1. β-Klotho Gene-Activated Scaffold Transiently Enhances Human ADSCs’ Stemness and
Pro-Reparative Genes
Gene expression analysis first showed that the ADSCs on the gene-activated scaffold
overexpressed the
β
-Klotho gene over 172-fold (p< 0.01) than the gene-free scaffold group
on day 3 (Figure 1A). This finding confirmed the efficient interaction of the ADSCs with the
gene-activated scaffold. Analysis of the proliferation marker Ki-67 further confirmed that
the ADSCs on the gene-activated scaffold also maintained a robust proliferative capacity
over 14 days (Figure 1A).
Having confirmed the overexpression of the anti-aging
β
-Klotho gene, we then as-
sessed stem cells’ rejuvenation through the activation of “pluripotency” factors in the
ADSCs. Overall, we noted a transient regulation of all the three transcription factors over
the 14 days (Figure 1B). However, the ADSCs only showed a significant increase in the
expression of the Oct-4 gene that sustained until day 14. Specifically, the ADSCs showed a
2.5-fold (p< 0.05) increase in the expression of the Oct-4 gene on day 3 before subsiding to
1.6-fold (p< 0.05) on day 14 relative to the gene-free scaffold group.
The next set of genes we investigated were the transforming growth factors beta 1
and 3, which are crucial for regulating scarless wound healing (Figure 1C). The levels
of pro-fibrotic TGF-
β
1 gene in the
β
-Klotho overexpressing ADSCs were found to be in
par with that of the gene-free scaffold group over the 14 days. However, the levels of
anti-fibrotic TGF-
β
3 were significantly (3.6-fold, p< 0.05) elevated during the early days
(day 3) and, as anticipated, subsided by about 0.4-fold by day 14, showing a controlled
expression of the regulatory gene.
2.2. β-Klotho Gene-Activated Scaffold Enhances the Paracrine Potency of Human ADSCs
2.2.1. Pro-Angiogenic Bioactivity
The bioactivity test of the CM generated by ADSCs-laden gene-activated scaffold first
showed that it could temporally control the metabolic activity in HUVECs. Day 3 CM
significantly enhanced the metabolic activity in HUVECs, while the use of aged CM from
day 14 significantly reduced the HUVEC’s metabolic activity (Figure 2A). Having observed
this, we then tested the pro-angiogenic impact of the CM from day 3 and 14 on the HUVECs
seeded on Matrigel. As anticipated, day 3 CM from the gene-activated scaffold group
significantly (p< 0.05) enhanced HUVEC tubule formation capacity and their branching
(Figure 2B). However, no further rise in the angiogenic activity occurred with day 14 CM
compared to that of the day 3 CM. Also, the day 14 CM from both the groups exhibited
similar pro-angiogenic efficiency (Figure 2C). Collectively, our finding indicates that the
gene-activated scaffold transiently expedites ADSCs’ pro-angiogenic response.
Pharmaceuticals 2021,14, 1168 4 of 16
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 4 of 17
Figure 1. Transcriptional activation of functional genes in the human ADSCs by β-Klotho gene-activated scaffold on day
3 (early) and day 14 (aged). (A) The ADSCs on the gene-activated scaffold showed a transient, high-level overexpression
of the therapeutic transgene β-Klotho until 14 days. Ki-67 marker indicates that the ADSCs proliferated well within the
gene-activated scaffold. (B) β-Klotho overexpression significantly induced the activation of pluripotency factor Oct-4 in
the ADSCs while maintaining basal levels of Nanog and Sox-2. (C) Increased activation of the stemness in the ADSCs was
further associated with a robust early activation of the anti-fibrotic gene TGF-β3, while maintaining basal levels of the
pro-fibrotic TGF-β1. ** and * indicates p < 0.01 and p < 0.05 respectively. Data represents mean ± standard deviation (n = 3).
Figure 1.
Transcriptional activation of functional genes in the human ADSCs by
β
-Klotho gene-activated scaffold on day
3 (early) and day 14 (aged). (
A
) The ADSCs on the gene-activated scaffold showed a transient, high-level overexpression
of the therapeutic transgene
β
-Klotho until 14 days. Ki-67 marker indicates that the ADSCs proliferated well within the
gene-activated scaffold. (
B
)
β
-Klotho overexpression significantly induced the activation of pluripotency factor Oct-4 in
the ADSCs while maintaining basal levels of Nanog and Sox-2. (
C
) Increased activation of the stemness in the ADSCs was
further associated with a robust early activation of the anti-fibrotic gene TGF-
β
3, while maintaining basal levels of the
pro-fibrotic TGF-
β
1. ** and * indicates p< 0.01 and p< 0.05 respectively. Data represents mean
±
standard deviation (n= 3).
Pharmaceuticals 2021,14, 1168 5 of 16
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 5 of 17
2.2. β-Klotho Gene-Activated Scaffold Enhances the Paracrine Potency of Human ADSCs
2.2.1. Pro-Angiogenic Bioactivity
The bioactivity test of the CM generated by ADSCs-laden gene-activated scaffold
first showed that it could temporally control the metabolic activity in HUVECs. Day 3
CM significantly enhanced the metabolic activity in HUVECs, while the use of aged CM
from day 14 significantly reduced the HUVEC’s metabolic activity (Figure 2A). Having
observed this, we then tested the pro-angiogenic impact of the CM from day 3 and 14 on
the HUVECs seeded on Matrigel. As anticipated, day 3 CM from the gene-activated
scaffold group significantly (p < 0.05) enhanced HUVEC tubule formation capacity and
their branching (Figure 2B). However, no further rise in the angiogenic activity occurred
with day 14 CM compared to that of the day 3 CM. Also, the day 14 CM from both the
groups exhibited similar pro-angiogenic efficiency (Figure 2C). Collectively, our finding
indicates that the gene-activated scaffold transiently expedites ADSCs’ pro-angiogenic
response.
Figure 2. Pro-angiogenic paracrine bioactivity of the ADSCs-laden gene-activated scaffold CM. (A) CM produced by the
ADSCs-laden gene-activated scaffold over the 14 days demonstrated an ability to temporally control HUVECs’ metabolic
activity. (B) Day 3 CM from the gene-activated scaffold group significantly enhanced endothelial tubule formation and its
branching compared to that of the gene-free scaffold group. (C) Tubule formation and branching of endothelial cells 6 h
post-exposure to CM from the ADSCs seeded gene-activated and gene-free scaffold. * indicates p < 0.05. Data represents
mean ± standard deviation (n = 3).
2.2.2. Dermal Fibroblast Healing and Maturation
One of the findings from the angiogenesis study is that as the ADSCs-laden
gene-activated scaffold matures, it no longer promotes angiogenesis. Furthermore, on
day 14, their angiogenic potency was similar to that of the gene-free scaffold group
(Figure 2B,C). Therefore, we investigated the influence of aged day 14 CM on dermal
fibroblast wound closure. Similar to the angiogenesis study result, both groups
demonstrated similar levels of fibroblast wound closure with day 14 CM. Specifically,
both the groups healed the wound by approximately 40% in 12 h (Figure 3A). This
finding revealed that during maturation, the gene-activated scaffold group supports but
does not promote dermal fibroblasts’ wound closure.
Figure 2.
Pro-angiogenic paracrine bioactivity of the ADSCs-laden gene-activated scaffold CM. (
A
) CM produced by the
ADSCs-laden gene-activated scaffold over the 14 days demonstrated an ability to temporally control HUVECs’ metabolic
activity. (B) Day 3 CM from the gene-activated scaffold group significantly enhanced endothelial tubule formation and its
branching compared to that of the gene-free scaffold group. (
C
) Tubule formation and branching of endothelial cells 6 h
post-exposure to CM from the ADSCs seeded gene-activated and gene-free scaffold. * indicates p< 0.05. Data represents
mean ±standard deviation (n= 3).
2.2.2. Dermal Fibroblast Healing and Maturation
One of the findings from the angiogenesis study is that as the ADSCs-laden gene-
activated scaffold matures, it no longer promotes angiogenesis. Furthermore, on day 14,
their angiogenic potency was similar to that of the gene-free scaffold group
(Figure 2B,C).
Therefore, we investigated the influence of aged day 14 CM on dermal fibroblast wound
closure. Similar to the angiogenesis study result, both groups demonstrated similar levels
of fibroblast wound closure with day 14 CM. Specifically, both the groups healed the
wound by approximately 40% in 12 h (Figure 3A). This finding revealed that during
maturation, the gene-activated scaffold group supports but does not promote dermal
fibroblasts’ wound closure.
Nevertheless, as the fibroblasts were stimulated with aged CM, we studied if the
CM influences the expression of matrix proteins involved in wound maturation. As
anticipated, dermal fibroblasts stimulated with CM from the gene-free scaffold group
abundantly expressed the pro-fibrotic collagen I protein (Figure 3B). On the contrary,
fibroblasts stimulated with CM from the gene-activated scaffold group demonstrated 50%
lower expression (p< 0.05) of the collagen I protein. Meanwhile, both the groups lacked
the expression of anti-fibrotic collagen III, and no differences were observed between the
two groups. Taken together, it implies that the gene-activated scaffold enhances ADSCs’
wound modulatory response during maturation.
Pharmaceuticals 2021,14, 1168 6 of 16
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 6 of 17
Nevertheless, as the fibroblasts were stimulated with aged CM, we studied if the
CM influences the expression of matrix proteins involved in wound maturation. As
anticipated, dermal fibroblasts stimulated with CM from the gene-free scaffold group
abundantly expressed the pro-fibrotic collagen I protein (Figure 3B). On the contrary,
fibroblasts stimulated with CM from the gene-activated scaffold group demonstrated
50% lower expression (p < 0.05) of the collagen I protein. Meanwhile, both the groups
lacked the expression of anti-fibrotic collagen III, and no differences were observed
between the two groups. Taken together, it implies that the gene-activated scaffold
enhances ADSCs’ wound modulatory response during maturation.
Figure 3. Paracrine influence on dermal healing and maturation. (A) Aged day 14 CM from both the groups exerted
similar levels of wound closure activity in human adult dermal fibroblasts by approximately 40% in 12 h. (B) CM from the
gene-activated scaffold significantly reduced the expression of pro-fibrotic collagen I in the fibroblasts. * indicates p < 0.05.
Data represents mean ± standard deviation (n = 3). In Figure 3B, GAS and GFS stand for gene-activated scaffold and
gene-free scaffold respectively.
2.3. β-Klotho Gene-Activated Scaffold Enhances Basement Membrane Regeneration with
Improved Anti-Fibrotic Response in Human ADSCs
Having observed the gene-activated scaffold’s controlled stimulation of ADSCs’
functionality, we ultimately investigated the ADSCs’ response towards the regeneration
of dermal matrices and proteins. The gene-activated scaffold robustly enhanced the
regeneration of ADSCs’ basement membrane. Specifically, the gene-activated scaffold
significantly promoted the deposition of basement membrane components laminin and
collagen IV (Figure 4A). After the semi-quantitative analysis, we also noted that the
matrix deposition followed a trend relative to the gene-free scaffold group. The trend as
observed was fibronectin (1.0-fold) < laminin (2.1-fold, p < 0.05) < collagen IV (8.8-fold, p <
0.01).
Once the ability to regenerate the basement membrane was determined, we then
assessed the expression of proteins crucial for superior qualitative healing, such as
reduced scarring. The ADSCs on the gene-activated scaffold demonstrated a significantly
(p < 0.05) reduced expression of the scar-associated contractile protein α-SMA. The
α-SMA expression in the ADSCs was lower by 50% relative to the gene-free scaffold
group (Figure 4B). Lastly, having observed the reduced α-SMA expression, we assessed
Figure 3.
Paracrine influence on dermal healing and maturation. (
A
) Aged day 14 CM from both the groups exerted
similar levels of wound closure activity in human adult dermal fibroblasts by approximately 40% in 12 h. (
B
) CM from the
gene-activated scaffold significantly reduced the expression of pro-fibrotic collagen I in the fibroblasts. * indicates
p< 0.05.
Data represents mean
±
standard deviation (n= 3). In Figure 3B, GAS and GFS stand for gene-activated scaffold and
gene-free scaffold respectively.
2.3.
β
-Klotho Gene-Activated Scaffold Enhances Basement Membrane Regeneration with Improved
Anti-Fibrotic Response in Human ADSCs
Having observed the gene-activated scaffold’s controlled stimulation of ADSCs’ func-
tionality, we ultimately investigated the ADSCs’ response towards the regeneration of
dermal matrices and proteins. The gene-activated scaffold robustly enhanced the regenera-
tion of ADSCs’ basement membrane. Specifically, the gene-activated scaffold significantly
promoted the deposition of basement membrane components laminin and collagen IV
(Figure 4A). After the semi-quantitative analysis, we also noted that the matrix deposi-
tion followed a trend relative to the gene-free scaffold group. The trend as observed was
fibronectin (1.0-fold) < laminin (2.1-fold, p< 0.05) < collagen IV (8.8-fold, p< 0.01).
Once the ability to regenerate the basement membrane was determined, we then
assessed the expression of proteins crucial for superior qualitative healing, such as reduced
scarring. The ADSCs on the gene-activated scaffold demonstrated a significantly
(p< 0.05)
reduced expression of the scar-associated contractile protein
α
-SMA. The
α
-SMA expression
in the ADSCs was lower by 50% relative to the gene-free scaffold group (Figure 4B). Lastly,
having observed the reduced
α
-SMA expression, we assessed the expression of the elastic
matrix protein elastin. The ADSCs on the gene-activated scaffold demonstrated a relatively
superior qualitative elastin expression through the deposition of mature fibrous network
of elastin compared to the ADSCs on the gene-free scaffold (Figure 4B).
Pharmaceuticals 2021,14, 1168 7 of 16
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 7 of 17
the expression of the elastic matrix protein elastin. The ADSCs on the gene-activated
scaffold demonstrated a relatively superior qualitative elastin expression through the
deposition of mature fibrous network of elastin compared to the ADSCs on the gene-free
scaffold (Figure 4B).
Figure 4. Deposition of pro-wound healing matrix proteins by the ADSCs in the gene-activated scaffold on day 14. (A)
The ADSCs in the gene-activated scaffold demonstrated significant regeneration of the basement membrane compared to
the ADSCs in the gene-free scaffold. The ADSCs predominantly deposited a relatively mature network of collagen IV
Figure 4.
Deposition of pro-wound healing matrix proteins by the ADSCs in the gene-activated
scaffold on day 14. (
A
) The ADSCs in the gene-activated scaffold demonstrated significant regen-
eration of the basement membrane compared to the ADSCs in the gene-free scaffold. The ADSCs
predominantly deposited a relatively mature network of collagen IV proteins, followed by laminin
and fibronectin. (
B
) The ADSCs in the gene-activated scaffold also expressed 2-fold lower of the
α
-SMA protein compared to the ADSCs in the gene-free scaffold. In conjunction with basement mem-
brane regeneration, the ADSCs in the gene-activated scaffold deposited qualitatively better elastin
matrix. All the images were captured through 20
×
objective using an IX73 Olympus microscope.
** and * indicates p< 0.01 and p< 0.05 respectively. Scale bar 50
µ
m. Data represents mean
±
standard
deviation (n= 3). α-SMA and fibronectin was double-immunostained.
Pharmaceuticals 2021,14, 1168 8 of 16
3. Discussion
This study aimed to investigate the functional impact of an anti-aging
β
-Klotho gene-
activated scaffold on human ADSCs for enhanced wound healing applications. Overall,
we found that the gene-activated scaffold offers controlled activation of ADSCs’ regenera-
tive abilities as depicted in Figure 5. Specifically, the gene-activated scaffold transiently
enhanced ADSCs’ stemness through the activation of transcription factor Oct-4. The
gene-activated scaffold also promoted early activation of the anti-fibrotic gene TGF-
β
3,
a crucial factor for controlled healing with reduced scarring. Furthermore, the activated
ADSCs temporally controlled endothelial angiogenesis and supported dermal fibroblasts’
healing through paracrine signaling. Towards maturation, the ADSCs also controlled the
activation of pro-fibrotic response in the dermal fibroblasts. Meanwhile, the ADSCs on
the gene-activated scaffold effectively regenerated the dermal basement membrane by
enhancing laminin and collagen IV deposition. This response was further associated with
reduced scar-associated
α
-SMA protein expression and improved qualitative elastin matrix
deposition. Our study collectively 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.
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 8 of 17
proteins, followed by laminin and fibronectin. (B) The ADSCs in the gene-activated scaffold also expressed 2-fold lower
of the α-SMA protein compared to the ADSCs in the gene-free scaffold. In conjunction with basement membrane
regeneration, the ADSCs in the gene-activated scaffold deposited qualitatively better elastin matrix. All the images were
captured through 20× objective using an IX73 Olympus microscope. ** and * indicates p < 0.01 and p < 0.05 respectively.
Scale bar 50μm. Data represents mean ± standard deviation (n = 3). α-SMA and fibronectin was double-immunostained.
3. Discussion
This study aimed to investigate the functional impact of an anti-aging β-Klotho
gene-activated scaffold on human ADSCs for enhanced wound healing applications.
Overall, we found that the gene-activated scaffold offers controlled activation of ADSCs’
regenerative abilities as depicted in Figure 5. Specifically, the gene-activated scaffold
transiently enhanced ADSCs’ stemness through the activation of transcription factor
Oct-4. The gene-activated scaffold also promoted early activation of the anti-fibrotic gene
TGF- β3, a crucial factor for controlled healing with reduced scarring. Furthermore, the
activated ADSCs temporally controlled endothelial angiogenesis and supported dermal
fibroblasts’ healing through paracrine signaling. Towards maturation, the ADSCs also
controlled the activation of pro-fibrotic response in the dermal fibroblasts. Meanwhile,
the ADSCs on the gene-activated scaffold effectively regenerated the dermal basement
membrane by enhancing laminin and collagen IV deposition. This response was further
associated with reduced scar-associated α-SMA protein expression and improved
qualitative elastin matrix deposition. Our study collectively 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.
Figure 5. A schematic of the functional impact of β-Klotho gene-activated scaffold on human ADSCs for wound healing
applications. The key findings of this study are the (1) transient enhancement of stem cell pluripotency and anti-fibrotic
response, (2) improved paracrine control towards angiogenesis, and pro-fibrotic collagen remodeling in dermal
fibroblasts, and ultimately, (3) increased maturation of the basement membrane with control over scar-associated
proteins’ expression. Dotted lines for elastin matrix imply improved qualitative deposition.
In wound healing strategies, plasmid-based gene therapy is applied to transiently
enhance cellular responses until wound repair is complete [37]. Our gene-activated
scaffold also shows that the therapeutic β-Klotho gene is transiently regulated over 14
days. The significantly enhanced (170-fold) expression of the β-Klotho mRNA at day 3
Figure 5.
A schematic of the functional impact of
β
-Klotho gene-activated scaffold on human ADSCs for wound healing
applications. The key findings of this study are the (1) transient enhancement of stem cell pluripotency and anti-fibrotic
response, (2) improved paracrine control towards angiogenesis, and pro-fibrotic collagen remodeling in dermal fibroblasts,
and ultimately, (3) increased maturation of the basement membrane with control over scar-associated proteins’ expression.
Dotted lines for elastin matrix imply improved qualitative deposition.
In wound healing strategies, plasmid-based gene therapy is applied to transiently
enhance cellular responses until wound repair is complete [
37
]. Our gene-activated scaffold
also shows that the therapeutic
β
-Klotho gene is transiently regulated over 14 days. The
significantly enhanced (170-fold) expression of the
β
-Klotho mRNA at day 3 could be
attributed to the immediate-early CMV promoter that is known to enhance transcriptional
activation of the encoded transgene [
38
]. However, in the MSCs, the CMV could undergo
DNA methylation or histone deacetylation that can reduce the transgene expression [
39
].
Moreover, the plasmids generally are non-replicating episomes, limiting the transfer of
transgene to dividing cells, causing a decline in the transgene expression over time [
40
].
The presence of heterochromatic markers originating from the bacterial sequences of the
Pharmaceuticals 2021,14, 1168 9 of 16
plasmid could further repress transgene expression [
41
], overall contributing to a transient
gene expression.
One of the avenues pursued for the treatment of hard-to-heal wounds is the application
of cell-seeded bioactive scaffolds. One reason is their ability to promote rapid wound repair.
Metabolically active cells in the graft secrete paracrine factors and undergo differentiation,
orchestrating the complex signaling events in the wound environment [
42
]. Stem cells, in
particular, have a unique ability to sense external stimuli and modulate their response to
restore homeostasis [
43
]. However, stem cells lose their stemness when they are expanded
in vitro
to generate large cell numbers for the graft. Our study shows that the stemness
can be transiently enhanced using a gene-activated scaffold carrying an anti-aging gene
β
-Klotho. Specifically, we noted that the anti-aging gene-activated scaffold enhanced
the expression of the stemness gene Oct-4 in the ADSCs. The Oct-4 is one of the key
transcriptional factors in embryonic stem cells and is essential for controlled embryonic
development [
44
]. In the ADSCs, the activation of the Oct-4 gene can promote their
proliferation and differentiation potential [
45
]. Thus, the increased expression of Oct-4 may
facilitate rapid differentiation of the ADSCs on the gene-activated scaffold into host cells
and aid in the healing process.
Having observed the activation of Oct-4 gene in the ADSCs, we then assessed the
angiogenic potency of the ADSCs. Angiogenesis is crucial for efficient integration of the
graft with the host tissue [
46
]. Cells in the graft trigger angiogenesis through the secretion
of paracrine factors. Angiogenesis is also a crucial event for granulation tissue development
and its activity diminishes as the granulation tissue mature [
47
]. This programmed limita-
tion of the angiogenic activity at the healing’s later stages is essential for controlling scarring
and hypergranulation that can impede re-epithelialization [
48
]. Our finding also shows that
the ADSCs on the gene-activated scaffold can enhance endothelial sprouting and control
their growth through paracrine signaling. Additionally, the significant increases in the
endothelial cells’ metabolic activity and sprouting in response to day 3 CM demonstrate
the potential of the ADSCs-laden gene-activated scaffold to integrate with the host tissue
rapidly. Moreover, the use of stem cells CM is a commonly adopted cell-free approach to
enhance quality wound healing [
49
]. As a limitation, we acknowledge that adult dermal
endothelial cells would serve a better cell model for studying angiogenesis; however to
maintain consistency with our previous studies and the HUVECs being widely accepted as
the “gold-standard” cell candidate [50], we adopted the HUVECs angiogenesis assay.
An increase in fibroblasts’ remodeling activity is a general feature of granulation tissue
maturation [
51
]. During this stage, collagen III matrix is gradually replaced by stronger
collagen I fibers [
51
]. Collagen fibers then promote wound contraction [
52
], and wound
contraction is indispensable for complete wound closure [
53
]. However, an aggressive
sustained increase in collagen I deposition can increase scar formation [
54
]. Controlling
scarring is crucial as it can inhibit the development of other skin appendages such as hair
follicles, sebaceous glands, and sweat glands, for example, in burn wounds [
54
]. Our
finding demonstrates that the aged (day 14) CM from the gene-activated scaffold group
could control the expression of pro-fibrotic collagen I in the adult dermal fibroblasts. This
controlled effect towards the fibroblasts occurred without further influence towards the
fibroblast’s migration or their anti-fibrotic collagen III expression relative to the gene-free
scaffold group. Therefore, we suggest that the ADSCs-laden gene-activated scaffold may
offer better control in limiting scarring
in vivo
through controlled collagen I expression.
Li et al., have also shown that the ADSCs CM can significantly reduce collagen I expres-
sion in fibroblasts derived from hypertrophic scars [
55
]. Another study by Wang et al.,
demonstrated similar reduction in collagen I expression in keloid-derived fibroblasts [
56
],
collectively demonstrating the anti-fibrotic potential of the ADSCs’ CM.
One of the significant findings that we noted with the application of the
β
-Klotho
gene-activated scaffold is the enhanced regeneration of basement membrane in the ADSCs.
The regeneration of the basement membrane is essential for blood vessel maturation and
complete re-epithelialization [
57
,
58
]. Importantly, the trend in which the proteins laminin
Pharmaceuticals 2021,14, 1168 10 of 16
(2.1-fold) and collagen IV (8.8-fold) are upregulated further indicates increased maturation
of the basement membrane in the gene-activated scaffold group [
59
]. Besides acting as
an anti-aging factor [
60
], the beta klotho functions to enhance sensitiveness to fibroblast
growth factor (FGF) by FGF receptors [
61
]. Additionally, increase in FGF signaling has
been found to promote basement membrane maturation through the deposition of laminin
and collagen IV [
62
]. Therefore, considering beta klotho’s role as a co-receptor of FGF, the
increased basement maturation in the gene-activated scaffold group is potentially mediated
by increase in ADSCs’ FGF signaling.
The basement membrane component remarkably upregulated in the gene-activated
scaffold group is the collagen IV protein. The collagen IV protein represents 50% of all
basement membrane [
63
] and provides structural stability to the basement membrane [
59
].
However, aging significantly reduces collagen IV expression in the dermal-epidermal
junction [
58
,
64
] and the lack of collagen IV can promote scar pathogenesis [
65
]. Thus, our
results suggest that the increased basement membrane regenerative potency of the ADSCs-
laden gene-activated scaffold can significantly aid in enhanced healing in aged patients.
Other studies have also shown that the adipose tissue-derived ECM possess tremendous
therapeutic potential for skin repair [
66
]. Furthermore, tissue-engineered grafts rich in
laminin and collagen IV is also of interest for respiratory epithelium repair [67].
In addition to the pro-fibrotic control towards the fibroblasts, our study further shows
that the ADSCs in the gene-activated scaffold also express a 2-fold lower expression of the
scar-associated protein
α
-SMA [
68
]. Although we did not evaluate its impact
in vivo
, the
reduction in local
α
-SMA expression is crucial for improved qualitative healing [
69
]. Using
fibrotic agents such as bleomycin [
70
] could be one way to evaluate better the anti-fibrotic
properties of the ADSCs/gene-activated scaffold construct
in vitro
; however, our focus
was to establish the provisional therapeutic potential of the construct.
Another indicator that the ADSCs-laden gene-activated scaffold may improve qual-
itative healing is the deposition of fibrous elastin matrix instead of patchy extracellular
secretions in the gene-free scaffold group. Elastin deposition is one of the main activities
that drive fetal scarless wound healing [
71
]. However, adult skins lack the deposition of
elastin [
71
]. As such, tropoelastin, a precursor of elastin, is often incorporated into bio-
material scaffolds to control scarring in wound healing [
72
]. Taken together, our findings
imply that the ADSCs/
β
-Klotho gene-activated scaffold construct may confer a strong
anti-fibrotic response in vivo.
4. Materials and Methods
4.1. Preparation of Gene-Activated Scaffold
The gene-activated scaffold was developed using a 2-step process. Firstly, solid
porous collagen-chondroitin sulfate scaffolds were fabricated by freeze-drying slurry of
bovine tendon type 1 collagen and chondroitin-6-sulfate derived from shark cartilage
(Sigma, UK). An optimized freeze-drying process designed to create uniform pores were
used to produce the scaffolds [
73
]. Based on our published protocol [
74
], the freeze-
dried scaffolds were then dehydrothermally (DHT) treated at 105
◦
C under vacuum for
sterilization and mechanical improvement. The sterilized scaffolds were then chemically
cross-linked with 14 mM N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride
and 5.5 mM N-Hydroxysuccinimide (2.5 molar ratio of EDC/NHS) (Sigma, UK) solution
to further improve the mechanical stability [
75
]. The cross-linked scaffolds were then
washed with PBS (Gibco, Paisley, UK) to remove residual chemicals. Once the scaffolds
were ready, polyplexes at an N/P10 ratio (nitrogen to phosphate ratio) were prepared
by mixing a predetermined volume of branched polyethyleneimine (PEI) solution with
plasmid DNA (pDNA) encoding for the human beta-Klotho gene (
β
-Klotho), obtained
from SinoBiological, Beijing, China. The N/P10 ratio was chosen based on our previous
studies that found that polyplexes formulated at an N/P 10 ratio could effectively form
small, stable cationic nanoparticles with plasmids as large as GLuc (5.76 kb) [21,23]. Prior
to use, the plasmids were diluted in endotoxin free water to obtain a working concentration
Pharmaceuticals 2021,14, 1168 11 of 16
of 0.5
µ
g/
µ
L. The plasmid contains human enhanced immediate-early cytomegalovirus
(CMV3) promoter to promote high-level stable and transient expression of the encoded
gene in mammalian cells.
The plasmid/PEI mix was allowed to settle for 30 min to self-assemble into polyplex
nanoparticles. The nanoparticles were then soak-loaded onto the scaffolds by pipetting
equal volumes of the polyplex solution per side of the scaffold. A total of 2
µ
g pDNA was
used per scaffold to develop the gene-activated scaffold.
4.2. Cell Seeding on β-Klotho Gene-Activated Scaffold
Human ADSCs (iXCells Biotechnologies, San Diego, CA, USA) were expanded to
passage 4 in the ADSCs growth medium (Cat no. MD0003) supplied by the company. A
total of 5
×
10
5
ADSCs (2.5
×
10
5
per side) were then seeded per gene-free scaffold (control,
n= 3) or gene-activated scaffold (test, n= 3). After letting the cells settle for about 20 min,
2 mL of transfection media OptiMEM (Gibco, UK) was added, and the cellularized scaffolds
were incubated at 37
◦
C for 24 h. After the 24 h incubation, 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 it with equal volume of
fresh media. All CM were stored at −80 ◦C until analysis.
4.3. qRT-PCR Analyses to Determine β-Klotho Gene Overexpression and Activation of
Functional Genes
In order to determine transient regulation of the target genes, cells were harvested
at days 3 (early) and 14 (late) post-seeding on the gene-free or gene-activated scaffolds.
The cells were first lysed using the Qiazol lysis reagent (Qiagen, Germantown, MD, USA).
Chloroform was then added to separate the cell lysate into protein, DNA and RNA phases.
Using the RNeasy Kit (Qiagen, Manchester, UK), the RNA was extracted, and their quality
and quantity were determined using a Multiskan Go plate reader (Thermo Scientific, UK)
with the absorbance set at 260 nm. Genomic DNA was then removed by mixing the RNA
with a genomic DNA wipeout buffer (Qiagen, Manchester, UK) and heating to 42
◦
C for
2 min. Subsequently, reverse transcription was performed to prepare the cDNA. Duplicates
of cDNA per replicate (n= 3) were loaded into the qRT-PCR plates and then the assay was
run using the primers listed in Table 1. Fold change in mRNA expression relative to the
cells on gene-free scaffold was calculated using the 2
−∆∆CT
method from averages of three
replicates per group. Human GAPDH (Hs_GAPDH_1_SG, Cat. No. QT00079247) was
used as the housekeeping gene.
Table 1. List of functional genes associated with ADSCs’ growth and development.
Function Primer (Catalog No.) Encoded Gene
Activation and proliferation
Hs_KLB_4_SG
(QT02454977) Beta Klotho (β-Klotho)
Hs_MKI67_1_SG
(QT00014203) Marker of proliferation (Ki-67)
Stemness or pluripotency promoters
Hs_POU5 F1_1_SG
(QT00210840) Octamer-binding transcription factor 4 (Oct-4)
Hs_NANOG_1_SG
(QT01025850) Homeobox protein (Nanog)
Hs_SOX2_1_SG
(QT00237601) Sex determining region Y-box 2 (Sox-2)
Wound healing regulators
Hs_TGFB3_1_SG
(QT00001302) Transforming growth factor beta 3 (TGF-β3)
Hs_TGFB1_1_SG
(QT00000728) Transforming growth factor beta 1 (TGF-β1)
Pharmaceuticals 2021,14, 1168 12 of 16
4.4. Bioactivity Analyses of Secreted Factors from the ADSCs on
β
-Klotho Gene-Activated Scaffold
4.4.1. Pro-Angiogenic Bioactivity Analyses
Next, we studied the paracrine potency of the ADSCs on
β
-Klotho gene-activated
scaffold. As angiogenesis is crucial for graft integration, we first studied the angiogenic
impact of CM on human umbilical vein endothelial cells (HUVECs). The assessment
was conducted in two stages. Firstly, a temporal effect of the CM on the HUVECs was
determined by assaying the metabolic activity in the HUVECs incubated with the CM
collected from all the time points—day 3, 7, 10, and 14. Briefly, 1
×
10
4
HUVECs/well in a
96-well plate was incubated in the CM for 24 h and the response was assayed using the
standard MTS assay. Subsequently, CM from days 3 and 14 were used for Matrigel assay.
For the Matrigel assay, the HUVECs were seeded at a density of 3
×
10
4
cells/well of a
48-well plate pre-coated with 120
µ
L of Matrigel for 30 min at 37
◦
C. At 6 h post-exposure
to CM, images of the morphological changes in the endothelial cells were captured using
an inverted microscope (IX73, Olympus, Japan) and the mean number of tubules and
branching points were counted using the ImageJ software (ImageJ, National Institutes of
Health, Bethesda, MD, USA).
4.4.2. Dermal Fibroblasts Healing and Maturation Analyses
After the angiogenesis assay, we investigated the CM’s potency for dermal healing by
employing a scratch assay of human adult dermal fibroblasts (iXcells, Cat no. 10 HU-014).
For this study, we specifically chose the aged CM from day 14 to study its influence on
fibroblasts’ wound closure during maturation. A total of 1
×
10
4
cells/well in a 96-well
plate were seeded and incubated overnight in the fibroblast growth medium. The next
day, a wound was created on the monolayer by horizontally scratching a 200 uL pipette tip
across the well. The monolayer was then rinsed with PBS to remove any debris and fed with
the CM. Wound closure was recorded between 12–16 h after the scratch. Immunostaining
was used to study the expression of pro-fibrotic collagen I (1:100, Novusbio, UK) and
anti-fibrotic collagen III (1:100, Novusbio, UK) in the fibroblasts. The fibroblasts were
counterstained with rhodamine (1:800, Abcam, UK) for F-actin imaging. Immunostaining
was performed as described in Section 4.5, with the exception of tissue processing and
deparafinization. Both HUVECs and the fibroblasts were used at passage 4.
4.5. Immunofluorescence Imaging of Extracellular Matrix Proteins
After 14 days of culture, the cellularized gene-free or gene-activated scaffolds were
harvested for detection of matrix deposition using immunofluorescence. The processing of
the samples was performed as described previously. Briefly, 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 7-
µ
m 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
×
3) and blocked using 10% NGS (Normal Goat
Serum, Invitrogen, Rockford, IL, USA)/5% BSA/0.3 M Glycine (prepared in permeabilizing
solution) for 1 h. After blocking, the slides were rinsed in PBS and then incubated at 4
◦
C
overnight with the antibodies to target matrix proteins listed in Table 2.
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 fluorescence microscope (Olympus BX43, Japan) at 20
×
objective. Samples incubated
with only secondary antibodies were used as controls.
Pharmaceuticals 2021,14, 1168 13 of 16
Table 2. List of primary antibodies to extracellular matrix proteins involved in wound healing.
Functional Roles Primary Antibodies
(Catalog No.)
Dilutions in 1% BSA
Solution
Basement membrane proteins
Fibronectin
(ab2413, Abcam, UK) 1:200
Laminin
(ab11575, Abcam, UK) 1:200
Collagen IV
(ab6586, Abcam, UK) 1:200
Scar-associated contractile protein Alpha-smooth muscle actin
(ab7817, Abcam, UK) 1:100
Elastic matrix protein Elastin
(ab21607, Abcam, UK) 1:200
Image Quantification
ImageJ software (ImageJ, NIH, MD, 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
×
mean gray value) of the images was determined and
then normalized to the number of cells (DAPI counting) to give a final mean fluorescence
density per cell. An average was quantified from 8–10 random images per replicate,
with a minimum of three replicates per group. The averages obtained from the three
replicates/group were then used for measuring relative expression between the groups.
4.6. Statistical Analysis
All results are expressed as mean
±
standard deviation. Unpaired, two-tailed t-test
was generally used to calculate the statistical significance between groups, where p< 0.05
was considered to be significant.
5. Conclusions
This study showed that the anti-aging
β
-Klotho gene-activated scaffold offers con-
trolled activation of the regenerative capacity of ADSCs. The key findings of this study
are the (1) transient enhancement of stem cell pluripotency and anti-fibrotic response,
(2) improved paracrine control towards angiogenesis, and pro-fibrotic collagen remodeling
in dermal fibroblasts, and ultimately, (3) increased maturation of the basement membrane
with control over scar-associated proteins’ expression. Conclusively, these results suggest
that the ADSCs-laden
β
-Klotho gene-activated scaffold may possess great potential a
treating a wide range of dermal wounds.
Author Contributions:
Conceptualization, A.L.L. and M.B.K.; methodology, A.L.L. and M.B.K.;
software, A.L.L.; validation, A.L.L., F.J.O. and M.B.K.; formal analysis, A.L.L.; investigation, A.L.L.;
resources, A.L.L., F.J.O. and M.B.K.; data curation, A.L.L. and M.B.K.; writing—original draft prepara-
tion, A.L.L.; writing—review and editing, A.L.L., F.J.O. and M.B.K.; visualization, A.L.L.; supervision,
F.J.O. and M.B.K.; project administration, M.B.K.; funding acquisition, F.J.O. and M.B.K. All authors
have read and agreed to the published version of the manuscript.
Funding:
This work was funded by RCSI Bahrain Internal Research Grant BR00090. The APC was
funded by RCSI Bahrain. F.O.B acknowledges funding from Science Foundation Ireland under
the M-ERA.NET program (Dressing4 Scars (16/M-ERA/3420) and the Advanced Materials and
BioEngineering Research Centre (AMBER; grants 12/RC/2278 and 12/RC/2278_P2) in addition to
EU BlueHuman Interreg Atlantic Area Project (grant EAPA_151/2016).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Pharmaceuticals 2021,14, 1168 14 of 16
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to privacy or ethical restrictions.
Conflicts of Interest: The authors declare that no conflict of interest exists.
References
1.
Laiva, A.L.; O’Brien, F.J.; Keogh, M.B. Innovations in gene and growth factor delivery systems for diabetic wound healing.
J. Tissue Eng. Regen. Med. 2018,12, e296–e312. [CrossRef] [PubMed]
2.
Makrantonaki, E.; Wlaschek, M.; Scharffetter-Kochanek, K. Pathogenesis of wound healing disorders in the elderly. JDDG J.
Dtsch. Dermatol. Ges. 2017,15, 255–275. [CrossRef] [PubMed]
3.
Ganguly, P.; El-Jawhari, J.J.; Burska, A.N.; Ponchel, F.; Giannoudis, P.V.; Jones, E.A. The analysis of
in vivo
aging in human bone
marrow mesenchymal stromal cells using colony-forming unit-fibroblast assay and the CD45lowCD271+ phenotype. Stem Cells
Int. 2019,2019, 5197983. [CrossRef]
4.
Westerweel, P.E.; Teraa, M.; Rafii, S.; Jaspers, J.E.; White, I.A.; Hooper, A.T.; Verhaar, M.C. Impaired endothelial progenitor cell
mobilization and dysfunctional bone marrow stroma in diabetes mellitus. PLoS ONE 2013,8, e60357. [CrossRef]
5.
Murray, R.Z.; West, Z.E.; Cowin, A.J.; Farrugia, B.L. Development and use of biomaterials as wound healing therapies. Burn.
Trauma 2019, 7. [CrossRef] [PubMed]
6.
Ebrahimian, T.G.; Pouzoulet, F.; Squiban, C.; Buard, V.; André, M.; Cousin, B.; Tamarat, R. Cell therapy based on adipose
tissue-derived stromal cells promotes physiological and pathological wound healing. Arterioscler. Thromb. Vasc. Biol.
2009
,
29, 503–510. [CrossRef]
7.
Wu, Y.; Chen, L.; Scott, P.G.; Tredget, E.E. Mesenchymal stem cells enhance wound healing through differentiation and angiogen-
esis. Stem Cells 2007,25, 2648–2659. [CrossRef] [PubMed]
8.
Foubert, P.; Gonzalez, A.D.; Teodosescu, S.; Berard, F.; Doyle-Eisele, M.; Yekkala, K.; Fraser, J.K. Adipose-Derived Regenerative
Cell Therapy for Burn Wound Healing: A Comparison of Two Delivery Methods. Adv. Wound Care
2016
,5, 288–298. [CrossRef]
[PubMed]
9.
Assi, R.; Foster, T.R.; He, H.; Stamati, K.; Bai, H.; Huang, Y.; Dardik, A. Delivery of mesenchymal stem cells in biomimetic
engineered scaffolds promotes healing of diabetic ulcers. Regen. Med. 2016,11, 245–260. [CrossRef] [PubMed]
10.
Jiang, Y.; Chen, B.; Liu, Y.; Zhufu, Z.; Yan, X.; Hou, X.; Tan, Q. Effect of collagen scaffold with adipose-derived stromal vascular
fraction cells on diabetic wound healing: A study in a diabetic porcine model. Tissue Eng. Regen. Med.
2013
,10, 192–199.
[CrossRef]
11.
Falanga, V.; Sabolinski, M. A bilayered living skin construct (APLIGRAF
®
) accelerates complete closure of hard-to-heal venous
ulcers. Wound Repair Regen. 1999,7, 201–207. [CrossRef]
12.
Hart, C.E.; Loewen-Rodriguez, A.; Lessem, J. Dermagraft: Use in the treatment of chronic wounds. Adv. Wound Care.
2012
,
1, 138–141. [CrossRef] [PubMed]
13.
Dinh, T.L.; Veves, A. The efficacy of Apligraf in the treatment of diabetic foot ulcers. Plast. Reconstr. Surg.
2006
,117, 152S–157S.
[CrossRef] [PubMed]
14.
Eaglstein, W.H.; Falanga, V. Tissue engineering and the development of Apligraf
®
, a human skin equivalent. Clin. Ther.
1997
,
19, 894–905. [CrossRef]
15.
Jiang, T.; Xu, G.; Wang, Q.; Yang, L.; Zheng, L.; Zhao, J.; Zhang, X.
In vitro
expansion impaired the stemness of early passage
mesenchymal stem cells for treatment of cartilage defects. Cell Death Dis 2017,8, e2851. [CrossRef] [PubMed]
16.
Liu, J.; Ding, Y.; Liu, Z.; Liang, X. Senescence in Mesenchymal Stem Cells: Functional Alterations, Molecular Mechanisms, and
Rejuvenation Strategies. Front. Cell Dev. Biol. 2020,8, 258. [CrossRef] [PubMed]
17.
Wang, W.; Xu, X.; Li, Z.; Lendlein, A.; Ma, N. Genetic engineering of mesenchymal stem cells by non-viral gene delivery. Clin.
Hemorheol. Microcirc. 2014,58, 19–48. [CrossRef]
18.
Deveza, L.; Choi, J.; Lee, J.; Huang, N.; Cooke, J.; Yang, F. Polymer-DNA Nanoparticle-Induced CXCR4 Overexpression Improves
Stem Cell Engraftment and Tissue Regeneration in a Mouse Hindlimb Ischemia Model. Theranostics
2016
,6, 1176–1189. [CrossRef]
[PubMed]
19. O’Brien, F.J. Biomaterials & scaffolds for tissue engineering. Mater. Today 2011,14, 88–95.
20.
Yannas, I.; Tzeranis, D.; Harley, B.; So, P. Biologically active collagen-based scaffolds: Advances in processing and characterization.
Philos. Trans. R. Soc. A Math. Phys. Eng. Sci. 2010,368, 2123–2139. [CrossRef]
21.
Laiva, A.L.; Raftery, R.M.; Keogh, M.B.; O’Brien, F.J. Pro-angiogenic impact of SDF-1
α
gene-activated collagen-based scaffolds in
stem cell driven angiogenesis. Int. J. Pharm. 2018,544, 372–379. [CrossRef] [PubMed]
22.
Lackington, W.A.; Raftery, R.M.; O’Brien, F.J.
In vitro
efficacy 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, 115–128. [CrossRef]
23.
Tierney, E.G.; Duffy, G.P.; Hibbitts, A.J.; Cryan, S.A.; O’Brien, F.J. The development of non-viral gene-activated matrices for
bone regeneration using polyethyleneimine (PEI) and collagen-based scaffolds. J. Control. Release
2012
,158, 304–311. [CrossRef]
[PubMed]
24.
Laiva, A.L.; O’Brien, F.J.; Keogh, M.B. SDF-1
α
gene-activated collagen scaffold drives functional differentiation of human
Schwann cells for wound healing applications. Biotechnol. Bioeng. 2021,118, 725–736. [CrossRef] [PubMed]
Pharmaceuticals 2021,14, 1168 15 of 16
25.
Laiva, A.L.; O’Brien, F.J.; Keogh, M.B. SDF-1
α
gene-activated collagen scaffold enhances provasculogenic response in a coculture
of human endothelial cells with human adipose-derived stromal cells. J. Mater. Sci. Mater. Med. 2021,32, 26. [CrossRef]
26.
Bonadio, J.; Smiley, E.; Patil, P.; Goldstein, S. Localized, direct plasmid gene delivery
in vivo
: Prolonged therapy results in
reproducible tissue regeneration. Nat Med 1999,5, 753–759. [CrossRef] [PubMed]
27.
Kolakshyapati, P.; Li, X.; Chen, C.; Zhang, M.; Tan, W.; Ma, L.; Gao, C. Gene-activated matrix/bone marrow-derived mesenchymal
stem cells constructs regenerate sweat glands-like structure in vivo. Sci. Rep. 2017,7, 17630. [CrossRef]
28.
Marolt, D.; Knezevic, M.; Vunjak-Novakovic, G. Bone tissue engineering with human stem cells. Stem Cell Res. Ther.
2010
,1, 10.
[CrossRef]
29.
Holm, J.S.; Toyserkani, N.M.; Sorensen, J.A. Adipose-derived stem cells for treatment of chronic ulcers: Current status. Stem Cell
Res. Ther. 2018,9, 142. [CrossRef]
30.
Laiva, A.L.; O’Brien, F.J.; Keogh, M.B. SDF-1
α
Gene-Activated Collagen Scaffold Restores Pro-Angiogenic Wound Healing
Features in Human Diabetic Adipose-Derived Stem Cells. Biomedicines 2021,9, 160. [CrossRef] [PubMed]
31.
Wan Safwani, W.K.; Makpol, S.; Sathapan, S.; Chua, K.H. The changes of stemness biomarkers expression in human adipose-
derived stem cells during long-term manipulation. Biotechnol. Appl. Biochem. 2011,58, 261–270. [CrossRef] [PubMed]
32.
Suku, M.; Laiva, A.L.; O’Brien, F.J.; Keogh, M.B. Anti-Ageing Protein
β
-Klotho Rejuvenates Diabetic Stem Cells for Improved
Gene-Activated Scaffold Based Wound Healing. J. Pers. Med. 2020,11, 4. [CrossRef]
33.
Ullah, M.; Sun, Z. Klotho Deficiency Accelerates Stem Cells Aging by Impairing Telomerase Activity. J. Gerontol. A Biol. Sci. Med.
Sci. 2019,74, 1396–1407. [CrossRef] [PubMed]
34.
Fan, J.; Sun, Z. The Antiaging Gene Klotho Regulates Proliferation and Differentiation of Adipose-Derived Stem Cells. Stem Cells
2016,34, 1615–1625. [CrossRef] [PubMed]
35.
Fan, F.; Li, Y.; Liu, Y.; Shao, L.; Yu, J.; Li, Z. Overexpression of klotho in adipose-derived stem cells protects against UVB-induced
photoaging in co-cultured human fibroblasts. Mol. Med. Rep. 2018,18, 5473–5480. [CrossRef] [PubMed]
36.
Zhang, F.; Wan, X.; Cao, Y.Z.; Sun, D.; Cao, C.C. Klotho gene-modified BMSCs showed elevated antifibrotic effects by inhibiting
the Wnt/β-catenin pathway in kidneys after acute injury. Cell Biol. Int. 2018,42, 1670–1679. [CrossRef]
37.
Branski, L.; Pereira, C.; Herndon, D.; Jeschke, M. Gene therapy in wound healing: Present status and future directions. Gene Ther.
2007,14, 1–10. [CrossRef] [PubMed]
38.
Radhakrishnan, P.; Basma, H.; Klinkebiel, D.; Christman, J.; Cheng, P.-W. Cell type-specific activation of the cytomegalovirus
promoter by dimethylsulfoxide and 5-aza-2’-deoxycytidine. Int. J. Biochem. Cell Biol. 2008,40, 1944–1955. [CrossRef] [PubMed]
39.
Wen, S.; Zhang, H.; Li, Y.; Wang, N.; Zhang, W.; Yang, K.; Wu, N.; Chen, X.; Deng, F.; Liao, Z.; et al. Characterization of constitutive
promoters for piggyBac transposon-mediated stable transgene expression in mesenchymal stem cells (MSCs). PLoS ONE
2014
,
9, e94397. [CrossRef] [PubMed]
40.
Hardee, C.L.; Arévalo-Soliz, L.M.; Hornstein, B.D.; Zechiedrich, L. Advances in non-viral DNA vectors for gene therapy. Genes
2017,8, 65. [CrossRef]
41.
Alhaji, S.Y.; Ngai, S.C.; Abdullah, S. Silencing of transgene expression in mammalian cells by DNA methylation and histone
modifications in gene therapy perspective. Biotechnol. Genet. Eng. Rev. 2019,35, 1–25. [CrossRef]
42.
Shevchenko, R.V.; James, S.L.; James, S.E. A review of tissue-engineered skin bioconstructs available for skin reconstruction. J. R.
Soc. Interface 2010,7, 229–258. [CrossRef] [PubMed]
43.
Ellison, D.D.; Suhail, Y.; Afzal, J.; Woo, L.; Kilic, O.; Spees, J.; Levchenko, A. Dynamic secretome of bone marrow-derived stromal
cells reveals a cardioprotective biochemical cocktail. Proc. Natl. Acad. Sci. USA 2019,116, 14374–14383.
44.
Shi, G.; Jin, Y. Role of Oct4 in maintaining and regaining stem cell pluripotency. Stem Cell Res. Ther.
2010
,1, 39. [CrossRef]
[PubMed]
45.
Han, S.-M.; Han, S.-H.; Coh, Y.-R.; Jang, G.; Ra, J.C.; Kang, S.-K.; Lee, H.-W.; Youn, H.-Y. Enhanced proliferation and differentiation
of Oct4-and Sox2-overexpressing human adipose tissue mesenchymal stem cells. Exp. Mol. Med.
2014
,46, e101. [CrossRef]
[PubMed]
46.
Perry, L.; Landau, S.; Flugelman, M.Y.; Levenberg, S. Genetically engineered human muscle transplant enhances murine host
neovascularization and myogenesis. Commun. Biol. 2018,1, 161. [CrossRef] [PubMed]
47.
DiPietro, L.A. Angiogenesis and wound repair: When enough is enough. J. Leukoc. Biol.
2016
,100, 979–984. [CrossRef] [PubMed]
48.
Kornhaber, R.; Jaeger, M.; Harats, M.; Aviv, U.; Zerach, A.; Haik, J. Treatment of hypergranulation tissue in burn wounds with
topical steroid dressings: A case series. Int. Med. Case Rep. J. 2016,9, 241–245. [CrossRef] [PubMed]
49.
Joseph, A.; Baiju, I.; Bhat, I.A.; Pandey, S.; Bharti, M.; Verma, M.; Singh, A.P.; Ansari, M.M.; Chandra, V.; Saikumar, G.; et al.
Mesenchymal stem cell-conditioned media: A novel alternative of stem cell therapy for quality wound healing. J. Cell. Physiol.
2020,235, 5555–5569. [CrossRef]
50.
Ni, C.-W.; Kumar, S.; Ankeny, C.J.; Jo, H. Development of immortalized mouse aortic endothelial cell lines. Vasc. Cell
2014
,6, 7.
[CrossRef] [PubMed]
51. Reinke, J.; Sorg, H. Wound repair and regeneration. Eur. Surg. Res. 2012,49, 35–43. [CrossRef] [PubMed]
52.
Brauer, E.; Lippens, E.; Klein, O.; Nebrich, G.; Schreivogel, S.; Korus, G.; Duda, G.N.; Petersen, A. Collagen Fibrils Mechanically
Contribute to Tissue Contraction in an In Vitro Wound Healing Scenario. Adv. Sci. 2019,6, 1801780. [CrossRef]
53.
Berry, D.P.; Harding, K.G.; Stanton, M.R.; Jasani, B.; Ehrlich, H.P. Human wound contraction: Collagen organization, fibroblasts,
and myofibroblasts. Plast Reconstr. Surg. 1998,102, 124–131. [CrossRef]
Pharmaceuticals 2021,14, 1168 16 of 16
54.
Marshall, C.D.; Hu, M.S.; Leavitt, T.; Barnes, L.A.; Lorenz, H.P.; Longaker, M.T. Cutaneous Scarring: Basic Science, Current
Treatments, and Future Directions. Adv. Wound Care 2018,7, 29–45. [CrossRef] [PubMed]
55.
Li, Y.; Zhang, W.; Gao, J.; Liu, J.; Wang, H.; Li, J.; Yang, X.; He, T.; Guan, H.; Zheng, Z.; et al. Adipose tissue-derived stem cells
suppress hypertrophic scar fibrosis via the p38/MAPK signaling pathway. Stem Cell Res. Ther.
2016
,7, 102. [CrossRef] [PubMed]
56.
Wang, X.; Ma, Y.; Gao, Z.; Yang, J. Human adipose-derived stem cells inhibit bioactivity of keloid fibroblasts. Stem Cell Res. Ther.
2018,9, 40. [CrossRef]
57.
Davis, G.E.; Senger, D.R. Endothelial extracellular matrix: Biosynthesis, remodeling, and functions during vascular morphogenesis
and neovessel stabilization. Circ. Res. 2005,97, 1093–1107. [CrossRef] [PubMed]
58.
Fisher, G.; Rittié, L. Restoration of the basement membrane after wounding: A hallmark of young human skin altered with aging.
J. Cell Commun. Signal. 2018,12, 401–411. [CrossRef] [PubMed]
59.
Hohenester, E.; Yurchenco, P.D. Laminins in basement membrane assembly. Cell Adhes. Migr.
2013
,7, 56–63. [CrossRef] [PubMed]
60.
Xu, L.; Idrees, M.; Joo, M.-D.; Sidrat, T.; Wei, Y.; Song, S.-H.; Lee, K.-L.; Kong, I.-K. Constitutive Expression of TERT Enhances
β
-Klotho Expression and Improves Age-Related Deterioration in Early Bovine Embryos. Int. J. Mol. Sci.
2021
,22, 5327. [CrossRef]
[PubMed]
61.
Suzuki, M.; Uehara, Y.; Motomura-Matsuzaka, K.; Oki, J.; Koyama, Y.; Kimura, M.; Asada, M.; Komi-Kuramochi, A.; Oka, S.;
Toru, T.
β
Klotho is required for fibroblast growth factor (FGF) 21 signaling through FGF receptor (FGFR) 1c and FGFR3c. Mol.
Endocrinol. 2008,22, 1006–1014. [CrossRef]
62.
Li, X.; Chen, Y.; Schéele, S.; Arman, E.; Haffner-Krausz, R.; Ekblom, P.; Lonai, P. Fibroblast growth factor signaling and basement
membrane assembly are connected during epithelial morphogenesis of the embryoid body. J. Cell Biol.
2001
,153, 811–822.
[CrossRef]
63.
LeBleu, V.S.; MacDonald, B.; Kalluri, R. Structure and function of basement membranes. Exp. Biol. Med.
2007
,232, 1121–1129.
[CrossRef]
64.
Roig-Rosello, E.; Rousselle, P. The Human Epidermal Basement Membrane: A Shaped and Cell Instructive Platform That Aging
Slowly Alters. Biomolecules 2020,10, 1607. [CrossRef] [PubMed]
65.
Yang, S.; Sun, Y.; Geng, Z.; Ma, K.; Sun, X.; Fu, X. Abnormalities in the basement membrane structure promote basal keratinocytes
in the epidermis of hypertrophic scars to adopt a proliferative phenotype. Int. J. Mol. Med.
2016
,37, 1263–1273. [CrossRef]
[PubMed]
66.
Zhou, Z.; Chen, Y.; Chai, M.; Tao, R.; Lei, Y.; Jia, Y.; Shu, J.; Ren, J.; Li, G.; Wei, W. Adipose extracellular matrix promotes skin
wound healing by inducing the differentiation of adipose-derived stem cells into fibroblasts. Int. J. Mol. Med.
2019
,43, 890–900.
[CrossRef]
67.
Hamilton, N.J.; Lee, D.D.H.; Gowers, K.H.; Butler, C.R.; Maughan, E.F.; Jevans, B.; Orr, J.C.; McCann, C.J.; Burns, A.J.;
MacNeil, S.; et al.
Bioengineered airway epithelial grafts with mucociliary function based on collagen IV-and laminin-containing
extracellular matrix scaffolds. Eur. Respir. J. 2020,55, 1901200. [CrossRef] [PubMed]
68.
Ibrahim, M.M.; Chen, L.; Bond, J.E.; Medina, M.A.; Ren, L.; Kokosis, G.; Selim, A.M.; Levinson, H. Myofibroblasts contribute to
but are not necessary for wound contraction. Lab. Investig. 2015,95, 1429–1438. [CrossRef] [PubMed]
69.
Zhang, Q.; Liu, L.N.; Yong, Q.; Deng, J.C.; Cao, W.G. Intralesional injection of adipose-derived stem cells reduces hypertrophic
scarring in a rabbit ear model. Stem Cell Res Ther 2015,6, 145. [CrossRef] [PubMed]
70.
Yoshizaki, A.; Yanaba, K.; Yoshizaki, A.; Iwata, Y.; Komura, K.; Ogawa, F.; Takenaka, M.; Shimizu, K.; Asano, Y.;
Hasegawa, M.; et al. Treatment with rapamycin prevents fibrosis in tight-skin and bleomycin-induced mouse models
of systemic sclerosis. Arthritis Rheum. 2010,62, 2476–2487. [CrossRef]
71.
Almine, J.F.; Wise, S.G.; Weiss, A.S. Elastin signaling in wound repair. Birth Defects Res. C Embryo Today
2012
,96, 248–257.
[CrossRef] [PubMed]
72.
Wen, Q.; Mithieux, S.M.; Weiss, A.S. Elastin Biomaterials in Dermal Repair. Trends Biotechnol.
2020
,38, 280–291. [CrossRef]
[PubMed]
73.
O’Brien, F.J.; Harley, B.A.; Yannas, I.V.; Gibson, L. Influence of freezing rate on pore structure in freeze-dried collagen-GAG
scaffolds. Biomaterials 2004,25, 1077–1086. [CrossRef]
74.
Haugh, M.G.; Jaasma, M.J.; O’Brien, F.J. The effect of dehydrothermal treatment on the mechanical and structural properties of
collagen-GAG scaffolds. J. Biomed. Mater. Res. Part A 2009,89, 363–369. [CrossRef]
75.
Haugh, M.G.; Murphy, C.M.; McKiernan, R.C.; Altenbuchner, C.; O’Brien, F.J. Crosslinking and mechanical properties significantly
influence cell attachment, proliferation, and migration within collagen glycosaminoglycan scaffolds. Tissue Eng. Part A
2011
,
17, 1201–1208. [CrossRef]
