Abstract
Objective
Autograft microskin transplantation has been widely used as a skin
graft therapy in full-thickness skin defect. However, skin grafting
failure can lead to a pathological delay wound healing due to a poor
vascularization bed. Considering the active role of adipose-derived
stem cell (ADSC) in promoting angiogenesis, we intend to investigate
the efficacy of autograft microskin combined with ADSC transplantation
for facilitating wound healing in a full-thickness skin defect mouse
model.
Material and methods
An in vivo full-thickness skin defect mouse model was used to evaluate
the contribution of transplantation microskin and ADSC in wound
healing. The angiogenesis was detected by immunohistochemistry
staining. In vitro paracrine signaling pathway was evaluated by protein
array and Gene Ontology, Kyoto Encyclopedia of Genes and Genomes
pathway, and protein-protein interaction network analysis.
Results
Co-transplantation of microskin and ADSC potentiated the wound healing
with better epithelization, smaller scar thickness, and higher
angiogenesis (CD31) in the subcutaneous layer. We found both EGF and
VEGF cytokines were secreted by microskin in vitro. Additionally,
secretome proteomic analysis in a co-culture system of microskin and
ADSC revealed that ADSC could secrete a wide range of important
molecules to form a reacting network with microskin, including VEGF,
IL-6, EGF, uPAR, MCP-3, G-CSF, and Tie-2, which most likely supported
the angiogenesis effect as observed.
Conclusion
Overall, we concluded that the use of ADSC partially modulates
microskin function and enhances wound healing by promoting angiogenesis
in a full-thickness skin defect mouse model.
Electronic supplementary material
The online version of this article (10.1186/s13287-019-1389-4) contains
supplementary material, which is available to authorized users.
Keywords: Adipose-derived stem cell, Microskin, Secretome,
Full-thickness skin defect, Wound healing
Background
Wound healing is a remarkably complex, and continuous process consisted
of hemostasis and coagulation, inflammation, proliferation, and wound
repairing with scar tissue formation [[43]1]. Inappropriate management
of wound care would result in a negative contribution to the healing
process and potential complications, such as delay or non-healing
wounds. Microskin grafting is a method of laying small sheets of the
skin graft on the cutaneous wound to enhance wound healing which has
been widely used as skin graft therapy in developing countries [[44]2].
The procedure is simple and economical which has been approved
successfully in full-thickness skin defect. However, there is a
limitation of microskin grafting such as lack of neovascularization,
keloid scar formation, and failure of transplantation due to poor wound
bed and ischemia-reperfusion (IR) [[45]3]. Therefore, burn surgeons
face a considerable challenge as to how to enhance the effectiveness of
microskin grafting.
In recent years, adipose-derived stem cell (ADSC) application, as a
stem cell-based therapy, has been proven to promote tissue regeneration
in chronic and non-healing wounds owing to their differentiation and
paracrine effects [[46]4]. ADSCs have attracted focus widely because
they can be harvested with minimal invasiveness. Early in vitro studies
reported that ADSCs could secrete various bioactive factors such as
vascular endothelial growth factor (VEGF), epidermal growth factor
(EGF), and hepatocyte growth factor (HGF), which were beneficial to
enhance endothelial cell (EC) function and promote angiogenesis
[[47]5]. Moreover, extracellular vesicles (EVs) released from ADSCs can
stimulate proliferation of human microvascular endothelial cells
(HMECs) and enhance pro-angiogenic function [[48]6]. These data show
the ability of ADSCs to enhance neovascularization, and paracrine
function may play a critical role in angiogenesis. In the model of
extended inferior epigastric artery skin flap in rats, treatment with
ADSCs increased flap survival by enhancing angiogenic response and
improving blood perfusion [[49]7]. Besides, significantly accelerate
neovascularization has been found in venous congested skin graft of
rabbit model treated with ADSCs [[50]8]. Owing to its paracrine
function, ADSCs have been widely applied as a new therapy to skin wound
healing and skin graft in recent years.
Although microskin grafting is the main method of massive skin defects,
there are still several problems we need to solve as previously
discussed, such as insufficient angiogenesis. We hypothesized that the
paracrine function of ADSCs might enhance angiogenesis promotion in
microskin grafting. Thus, we aim to explore if microskin in a
combination of ADSCs could promote the wound healing of full-thickness
skin defects and conquer the limitation of microskin grafting. In our
study, a number of cytokines secreted by the co-culture system of
microskin and ADSCs, including vascular endothelial growth factor
(VEGF), interleukin 6 (IL-6), epidermal growth factor (EGF), urokinase
plasminogen activator receptor (uPAR), monocyte chemotactic protein-3
(MCP-3), granulocyte colony-stimulating factor (G-CSF), and tyrosine
kinase with immunoglobulin-like and EGF-like domains 2 (Tie-2), were
identified by high-throughput protein array. These cytokines contribute
to angiogenesis and promote wound healing. Therefore, our in vivo and
in vitro study suggests that the combination of microskin and ADSCs
could be a promising therapy to promote wound healing of full-thickness
skin defect.
Materials and methods
Animals
The animal protocol was approved by the Institutional Animal Research
Committee Approval of Sun Yat-sen University. All the animals were
purchased from the Animal Center for Medical Experiment of Guangdong.
This study has been conducted under the guideline of the Guide for the
Care and Use of Laboratory Animals.
Isolation, culture, and characterization of adipose-derived stem cell
ADSC extraction was performed as described by Zuk et al. [[51]9]. The
inguinal subcutaneous fat was isolated from the Balb/c mice (male,
12 weeks old). The adipose was washed with phosphate-buffered saline
(PBS, Gibco, USA) consists of 1% penicillin-streptomycin Solution
(Keygen, Jiangsu, China) three times, minced with scissors into pieces
less than 1-mm diameter, washed with PBS, and centrifuge for 5 min at
400g three times. For the floating tissue, a threefold volume of 1%
type I collagenase (Gibco, USA) was added, and the admixture was then
digested in 37 °C water bath and shaken gently every 5 min for 45 min.
The deposit was suspended with culture medium and pass through a 70-μm
filter to removed undigested tissue. The pellets were resuspended with
culture medium to a final concentration of 5 × 10^6cells/ml; then, the
cells were placed in a 37 °C incubator supplied with 5% CO[2] and 95%
humidity. The medium was changed every 2 days. The third or fourth
passage cells were used for various experiments.
After the third or fourth passage, cells were harvested and applied to
characterize the CD markers of mesenchymal stem cells. The protocols
were adopted and followed by other previously published studies
[[52]10]. Briefly, 50 μl of cell suspension was incubated with a
fluorochrome-conjugated monoclonal antibody for 1 h in the dark at room
temperature, washed three times with PBS, and analyzed using a FACS
Calibur flow cytometer (Becton Dickinson, San Jose, CA). The antibodies
used in the experiments were HLA-DR, CD11b, CD19, CD34, CD45, CD73,
CD90, CD105 (Abcam, USA). Mouse fluorochrome-conjugated isotype control
IgG antibodies (Abcam, USA) were used in the experiments as a negative
labeling control.
To analyze cell differentiation abilities of adipogenic, osteogenesis,
and chondrogenic, ADSCs were cultured in adipogenic differentiation
medium for 2 weeks, osteogenesis differentiation medium for 3 weeks,
and chondrogenic differentiation medium for 4 weeks (ScienceCell, USA).
Cells were fixed with 4% paraformaldehyde in PBS for 1 h at room
temperature and stained with Oil Red O, Alizarin Red, and Alcian blue
(Sigma) solution. The results were observed under a phase contrast
microscope (Nikon, Japan).
Fabrication of microskin
Preparation of microskin was performed as described by Zhang et al.
[[53]11], with a bit of modification. Balb/c mice (male, 12 weeks old)
weighing approximately 22 g were used in this experiment. Their backs
were shaved and wiped with 75% ethyl alcohol after intraperitoneal
injection of 50 mg/kg sodium pentobarbital for anesthesia. Then, a
piece of full-thickness skin (2 cm × 3 cm) was removed from the
subjected mouse and cut into 5 mm × 5 mm with a scissor. The debris of
the skin was then immersed in PBS and washed three times. For the
debris of the skin, a threefold volume of 0.25% dispase was added and
incubated at 4 °C overnight to detach the dermis and the epidermis. The
epidermis was cut into less than 1-mm diameter (microskin) and washed
with PBS.
In vivo mouse wound healing model
Thirty Balb/c mice (male, 8 weeks old), weighing 22 ± 4 g, were used in
this experiment. Mice were housed in the environment without specific
pathogen and free to access standard food and water with 12-h
photoperiods. Before the surgical procedure, all the mice accepted
anesthesia by 50 mg/kg sodium pentobarbital by intraperitoneal
injection. Their dorsal surface, including the surgical area, was
shaved exhaustively and wiped with 75% ethyl alcohol twice. Mice were
randomized into two parts (n = 15 each) depending on the processing
treatment after surgery. Two round shape of the full-thickness wound
(1.2-cm diameter) were created in the middle of the back of each mouse.
The wounds were photographed by digital camera immediately. According
to local treatment used for each mouse, one wound was transplanted 1/4
area autologous microskin evenly (as previously described) and 30 μl
PBS (MS group), the other was transplanted 30 μl PBS as a negative
control (control group). A coupled mouse was manipulated similarly, and
one wound was transplanted 1/4 area autologous microskin and 30 μl ADSC
with 1 × 10^5 cells (MS+ADSC group), the other was transplant 30 μl
ADSC with 1 × 10^5 cells as control (ADSC group). The ADSCs were
prepared before the surgery and injected into the surface of the wound
area. A polyethylene collar was stitched to the adjacent skin to retain
the margin of the wound. The mice were administered with sodium
salicylate (150 mg/kg) for pain control and antibiotic for the
following 2 days. At the time point of 7 days and 14 days after
surgery, the wounded skin was photographed. To evaluate the therapeutic
effect of each treatment, we performed a photograph of the wounded skin
by a digital camera at 0 days, 7 days, and 14 days post-treatment and
analyze the data by ImageJ (NIH, Bethesda, MD) software. All the
measurements of the wound area and wound contraction were followed as
previous studies [[54]12]. For the wound area studies, we defined that
the actual wound area was open wound area and it was calculated using
the following formula:
[MATH: %wound area=W0−W1−W2/
W0×100%
:MATH]
For the wound contraction studies, it was calculated using the
following formula:
[MATH: %wound contraction=W0−W1/
W0×100%
:MATH]
where W[0] was the original wound area on day 0, W[1] was the open
wound area at day 7 and day 14, and W[2] was the area of microskin
adhering to the skin or re-epithelialization area. The sum of
contraction area, re-epithelialization area or microskin area, and open
wound areas equal 100% of the original wound size. Meanwhile, half of
the mice were sacrificed by CO[2] asphyxiation and carefully harvested
the wounded skin for histological analysis at each time point.
Histochemistry and immunohistochemistry
For histochemistry and immunohistochemistry analysis, the wounded skins
taken at each time point were excised and fixed in 4% paraformaldehyde,
embedded in paraffin, and sectioned vertically into 4-μm-thick
sections. For histological observations, representative sections were
stained for hematoxylin and eosin (H&E) following conventional
protocols. Alpha smooth muscle actin (a-SMA), CD31, and vascular
endothelial growth factor (VEGF) were chosen to perform
immunohistochemistry to evaluate fibrosis and neovascularization
following routine protocols [[55]13]. Briefly, the tissue sections were
performed in citrate-based antigen retrieval for 15 min and blocked
with normal goat serum for 30 min. Then, the sections were incubated
with anti-CD31 (1:100; Abcam, UK), anti-VEGF (1:100; Abcam, UK), and
anti-alpha smooth muscle actin (anti-α-SMA, 1:150; Abcam, UK)
antibodies at 4 °C overnight, separately. After washing with PBS, the
sections were developed with DAB and counterstained with hematoxylin.
The sections were analyzed and images acquired with an upright optical
microscope (Nikon, Japan). Neovascularization at the wound sites was
detected by CD31 staining. Microphotographs were captured, and
quantification of CD31-positive (+) blood vessels was performed in ten
random fields per section. Only the blood vessels, which have a
diameter of 2–10 μm, were counted as one vessel [[56]14]. For the
measurement of scar thickness (the distance from the epidermal-dermal
junction down to the panniculus carnosus, Additional file [57]4: Figure
S3A), five random distances with equal gap (1000 μm) of scar thickness
were measured in the wound area which are determined on three H&E
staining sections each group using ImageJ software [[58]15].
Interaction detection by co-culture of microskin and ADSC, ADSC, and
fibroblasts
It is reported that ADSCs could differentiate into keratinocyte and
endotheliocyte cells in vitro [[59]16, [60]17]. To investigate whether
ADSCs differentiated into keratinocyte or endotheliocyte cells while
co-culturing with microskin, we performed a co-culture system, which
was adopted and followed by previously published studies [[61]18]. An
8-μm micropore, 6-well Transwell plate (Millipore, USA) was used to
co-culture microskin and ADSC. 1 × 10^5cells/ml ADSC was seeded in the
lower chamber, and microskin about 1-cm^2 epidermis area was put in the
upper chamber. The Transwell system was supplied by DMEM medium with 2%
FBS. After incubated for 7 days and 14 days, total RNA and protein of
ADSC were extracted and preserved in − 80 °C for further research.
Cultured ADSC supplied by DMEM medium with 2% FBS was taken as the
control group. qRT-PCR and Western blot were applied to investigate the
protein and mRNA expression of keratin 5 (CK5), keratin 19 (CK19),
kinase insert domain receptor (KDR), and von Willebrand factor (VWF)
(Additional file [62]1: Table S1) in the co-culture system of MS and
ADSC.
Secretion function of microskin
To investigate the secretion ability of microskin, microskin about
1-cm^2 epidermis area was put in a 6-well plate uniformly and incubated
for 3 days, 7 days, and 14 days. The supernatant was taken for EGF and
VEGF secretion detection (Elabscience, China) by enzyme-linked
immunosorbent assay (ELISA). The concentration of cytokines
demonstrated the secretion function of microskin.
Paracrine analyze by protein array
The cultured microskin suspends and the co-cultured microskin with ADSC
suspend were taken as protein array samples. A total of 60 proteins
were selected, including growth factors, chemotactic factors, and
inflammation factors. All the samples were analyzed using an array
(RayBiotech, Norcross, GA, USA, GSH-ANG-1000). All experiments were
conducted according to the manufacturer’s instructions. Briefly, after
60 min of incubation with blocking buffer, 60 μl of 100-fold
concentrated samples was added to each well. After overnight incubation
at 4 °C and extensive washing, the biotin-labeled detection antibody
was added for 2 h and then washed away. AlexaFluor 555-conjugated
streptavidin was then added and incubated for 1 h at room temperature.
The signals (532-nm excitation, 635-nm emission) were scanned and
extracted using an InnoScan 300 scanner (Innopsys, Carbonne, France).
Raw data from the array scanner were provided as images (.tif files)
and spot intensities (tab-delimited.txt file) through Mapix 7.3.1
Software. All experiments were conducted according to the
manufacturer’s instructions. Individual array spots were
background-subtracted locally and normalized through two positive
controls. Calculate the mean signal-BKG for each set of duplicate
standards and samples. Then, plot the standard curve on log-log graph
paper, with standard concentration on the x-axis and signal-BKG on the
y-axis.
At last, draw the best-fit straight line through the standard points.
Concentrations of all serum proteins detected were determined according
to its standard curve. It was considered as differentially expressed
protein (DEP) by comparison of the signal values between the groups
based on p < .05 by t test, signal value > 150, and fold change
(FC) ≥ 1.2 or ≤ 0.83.
Gene Ontology, KEGG pathway analysis, and integration of protein-protein
interaction network analysis
We performed Gene Ontology (GO) analysis and the Kyoto Encyclopedia of
Genes and Genomes (KEGG) pathway to analyze the DEPs by using Online
String Tools. GO analysis was used to annotate genes and gene products
including cellular component, biological process, and molecular
function. KEGG was performed for systematic analysis of the pathways in
which genes in DEPs were involved in our study by R Bioconductor
package clusterProfiler [[63]19].
STRING version 11.0 is a database of protein-protein interactions
(PPIs) which covers 5090 organisms. The STRING database is performed to
access the protein-protein interactions including direct (physical) and
indirect (functional) associations [[64]20]. To evaluate the
interrelation among DPEs detected in this study, STRING was utilized
and obtained a PPI network through the function and pathway enrichment
analysis. It was considered statistically significant with p < .05.
Statistical analysis
Results from the quantitative studies of wound healing analysis in
vivo, Western blot, and qRT-PCR were expressed as the mean ± standard
deviation (SD). Results from the quantitative studies of the blood
vessels in IHC staining were expressed as the mean ± standard error of
the mean (SEM). Three independent experiments were performed for
validity, and at least three samples per each test were taken for
statistical analysis. Statistical comparisons between the two groups
were performed by two-tailed Student’s t test. Differences among
multiple groups were statistically analyzed using one-way analysis of
variance (ANOVA). Differences were considered significant when
p < 0.05.
Results
Characterization of ADSCs
According to the Mesenchymal and Tissue Stem Cell Committee of the
International Society for Cellular Therapy, we investigated the
expression levels of cell surface markers [[65]10]. Flow cytometry
analysis indicated that more than 98% of cultured cells expressed CD73
(99.5%), CD90 (98.7%), and CD105 (98.8%), whereas a small fraction of
them expressed HLA-DR (1.7%), CD45 (1.8%), CD34 (1.9%), CD19 (0.1%),
and CD11b (0.2%) (Additional file [66]2: Figure S1A). This expression
of cell surface markers is a characteristic protein expression of
ADSCs.
Differentiation abilities of the isolated cell were further examined by
adipogenic, osteogenesis, and chondrogenic. The isolated cells were
cultured in special supplemented medium of adipogenic, osteogenesis,
and chondrogenic. Staining of Oil Red O (Additional file [67]2: Figure
S1B), Alizarin Red (Additional file [68]2: Figure S1C), and Alcian blue
(Additional file [69]2: Figure S1D) was carried out to verify the
differentiation capacity. The mesenchymal phenotype was supported by
their multipotency. All these results demonstrated that the isolated
cells were ADSCs.
Autograft microskin combined with adipose-derived stem cell enhanced wound
healing in full-thickness skin defect mouse model
We originally designed to figure out the effect of microskin combined
with ADSCs on cutaneous wound healing in a full-thickness skin defect
mouse model. Full-thickness skin wounds were made on the mouse’s back
and photographed the open wound area immediately. After surgery,
different treatments were performed in the wound area. Besides, the
wound area was assessed by photo observation along the time of
transplantation and analyzed by photo analyzed software (Fig. [70]1a).
There are no apparent signs of infection of any groups throughout the
experiment, including no exudate or purulent drainage. On day 7
post-treatment, the wound area of the MS+ADSC group (34.16% ± 3.113%)
was smaller than all other groups (ADSC group, 58.67% ± 3.900%; MS
group, 49.55% ± 5.170%; and control groups, 68.05% ± 2.687%; all
p < .0001). At 14 days after treatment, the wound area of the MS+ADSC
group (3.514% ± 1.261%) was almost closed while the other groups (ADSC
group, 8.792% ± 0.743%; MS group, 7.039% ± 1.177%; all p < .001) still
not completely closed with an obviously non-healed area, especially in
the control group (11.09% ± 1.324%, p < .0001). (Fig. [71]1b, c). It
should be noted that the MS+ADSC group (56.27% ± 3.033%) could suppress
the wound contraction on day 14 post-treatment, compared to other
groups (ADSC group, 75.29% ± 3.679%; MS group, 73.93% ± 3.224%; and
control groups, 81.92% ± 2.380%; all p < .0001) (Fig. [72]1d). Besides,
a majority of microskin graft was survival in the newly formed skin
coverage close to normal skin in the MS+ADSC group. The newly formed
skin was flat without infection and open wound. Besides, there were no
signs of hypertrophic scarring, eschar, and hyperpigmentation in the
MS+ADSC group. On the contrary, we could observe an obvious wound
contraction and eschar in the ADSC group (Additional file [73]3: Figure
S2B), MS group (Additional file [74]3: Figure S2C), and control group
(Additional file [75]3: Figure S2D). It demonstrated that the treatment
of microskin and ADSCs could enhance full-thickness skin defect wound
healing in mouse model and suppress wound contraction.
Fig. 1.
[76]Fig. 1
[77]Open in a new tab
Treatment of ADSC+MS promoted wound healing in a full-thickness skin
defect mouse model. a Mice were randomized into two parts (n = 15 each
part). One group accepted the treatment of microskin or PBS, the other
accepted microskin plus ADSCs or ADSCs after surgery. Photograph of the
wound area was performed at days 0, 7, and 14, and mice were sacrificed
to have IHC procedure at days 7 and 14. b Representative of a
full-thickness wound of each group at day 0, and wound closure can be
observed at days 7 and 14 which the MS+ADSC group presented the most
remarkable effect of wound healing. c Quantitative evaluation of the
wound area on days 0, 7, and 14 post-treatment. ^###p < .001, the
MS+ADSC group compared to all other groups; ****p < .0001, the MS+ADSC
group compared to the ADSC group and control group; ***p < .001, the
MS+ADSC group compared to the MS group. d Quantitative evaluation of
wound contraction at day 14 post-treatment. ****p < .0001; **p < .01;
*p < .05, compared to the control group
In addition, we found a difference between the MS+ADSC group and MS
group in hematoxylin and eosin staining. In wound beds at day 14
post-surgery, a newly formed epithelium could be found in the MS+ADSC
group with stratum corneum. Besides, the newly formed epithelium of the
MS+ADSC group is thicker than the other groups including the MS group.
Especially, in the MS+ADSC group, the microskin probably had become
appendages of the freshly formed skin, and we seldom observed this
phenomenon in the MS group (Fig. [78]2a). In day 14 post-surgery, also,
the MS+ADSC group (0.8143 ± 0.10 mm) presented with a significantly
smaller scar thickness than the other groups (ADSC group,
1.00 ± 0.16 mm, p < .05; MS group, 1.05 ± 0.13 mm, p < .01; control
group, 1.24 ± 0.15 mm, p < .0001; Additional file [79]4: Figure S3).
Fig. 2.
[80]Fig. 2
[81]Open in a new tab
Microscopic appearance of wound beds post-surgery. a Hematoxylin and
eosin staining of wound beds at day 14 post-surgery. Wounds treated
with MS+ADSC showed a newly formed, hyperplastic epithelium that
covered the wound area. The black arrows indicate the microskin grafts
had become appendages of the newly formed skin. The asterisks indicated
normal adnexal structures. “N” is represented for normal skin, and “w”
is represented for wound area. b α-SMA staining of wound beds at day 14
post-treatment. At day 14, the expression of α-SMA in wound tissue was
decreased in the MS+ADSC group compared to others. Scale bars are 100
and 500 μm. c CD31 staining of the wound area at days 7 and 14 after
treatment. Black arrows indicate CD31-positive vessels. d the number of
CD31-positive (+) blood vessels per high-power fields (HPFs) (× 20)
were quantified to a particular time point. The data expressed are the
average means ± SEM, n = 5. ****p < .0001, compared to the ADSC group,
MS group, and control group; **p < .01; *p < .05; ns, not significant,
compared to the control group
Combination of ADSCs and microskin suppressed the expression of α-SMA and
increased the expression of CD31
IHC staining suggested that the expression of α-SMA in the MS+ADSC
group was significantly reduced compared to the MS group and control
group, which means the fibrosis in hypodermis was inhibited
(Fig. [82]2b). Neovascularization at the area of the wound was detected
by CD31 staining, which was the marker protein of endothelial cells.
The representative images of CD31 staining were presented (× 20), and
black arrows indicated CD31-positive vessels (Fig. [83]2c). At day 7,
as expectedly, it was shown that the number of new blood vessels in the
wound area treated with a combination of microskin and ADSCs
(16.27% ± 0.493%) was much higher than the other groups (ADSC group,
9.267% ± 0.431%; MS group, 10.11% ± 0.588%; and control group
7.889% ± 0.247%; Fig. [84]2d), while there was no significance between
the ADSC group and MS group. Although the number of new blood vessels
in the ADSC group and MS group was much more than the control group,
however, the neovascularization was uneven with a lower density,
compared to the MS+ADSC group. At day 14 after treatment, we could
observe a large number of mature blood vessel formation in the MS+ADSC
group (17.03% ± 0.606%) compared to the other groups (ADSC group,
10.30% ± 0.276%; MS group, 11.40% ± 0.423%; and control group,
9.667% ± 0.27%; Fig. [85]2d). Notably, there was no significance in
both the ADSC group and MS group compared to the control group. These
finding indicated that vascular regeneration was better in the MS+ADSC
group during full-thickness skin defect regeneration. Therefore, the
combination of microskin and ADSCs might accelerate skin wound healing
through suppressing fibrosis and promoting vascularization.
ADSCs did not differentiate into keratinocyte or endotheliocyte cells while
co-culturing with microskin
To investigate whether ADSCs differentiated into keratinocyte or
endotheliocyte cells while co-cultured with microskin, we designed a
co-culture system as previously described. Western blot and qRT-PCR
investigated the expression of CK5, CK19, KDR, and VWF of ADSC in the
co-culture system and control ADSC. The protein and mRNA expression of
CK5 and CK19 were both downregulated in 7 days and 14 days, along with
the downregulation of the protein and mRNA expression of KDR and VWF
(Fig. [86]3a, b). This data demonstrated that the function of ADSC in
the co-culture system was not differentiation.
Fig. 3.
[87]Fig. 3
[88]Open in a new tab
MS+ADSC may enhance wound healing through paracrine function rather
than the differentiation of ADSCs. a mRNA expression of CK5, CK19, KDR,
and VWF are shown in each group. b Representative Western blot bands
for CK5, CK19, VWF, and KDR expression are shown in each group
including at days 7 and 14 post-treatment. In co-cultured system, ADSCs
were all downregulated compared to the control group. c The
concentration of EGF and VEGF secreted by microskin kept stabilizing at
7 days and decreased at 14 days. Both at 7 and 14 days, EGF and VEGF
secreted by microskin were higher than the control group (FBS) which
demonstrated the microskin could release biological factor. *p < .05,
compared with the control group
To further identify the secretion ability of microskin, we performed a
tissue culture experiment [[89]21]. In our experiment, EGF and VEGF
were selected as a reference of secreted cytokines by microskin. At
3 days, 7 days, and 14 days, the secretion of EGF and VEGF was higher
than the control group (culture medium) (Fig. [90]3c). The secretion of
EGF and VEGF began declining since the seventh day of culture. These
data indicated microskin was able to secrete cytokines in vitro.
Protein array showed several pro-angiogenic cytokines secreted by a
co-culture system of ADSC and microskin
A total of 60 angiogenic cytokines were detected in the culture
supernatants of microskin (MS+A group), ADSCs (ADSC group), and
microskin (MS group) on day 7. As expected, all three groups highly
expressed several growth factors, including angiopoietin-1 (ANG-1),
placental growth factor (PIGF), Tie-2, hepatocyte growth factor (HGF),
EGF, and VEGF. Specifically, there were 31 common differential
cytokines detected in the MS+A group and were upregulated compared to
the MS group ADSC group (Fig. [91]4a). Also, the heat map of
differentially expressed proteins and the detailed cluster analysis
were presented (Fig. [92]4b, c). From the heat map, we could find that
co-culture of microskin and ADSCs could upregulate the expression of
most cytokines detected in vitro compared to culturing microskin and
ADSCs only. Generally, there were 29 and 32 upregulated DEPs detected
in the MS+ADSC group compared to the ADSC group and MS group,
respectively. Also, the top upregulation cytokines detected in the
MS+ADSC group were I-309, EGF, G-CSF, Tie-2, and VEGF-R2 in comparison
with the ADSC group; while it was Tie-2, MCP-3, HGF, ANG-1, and GSF in
comparison with the ADSC group. We could confirm that there was a
regulation of biological factors while co-culture of microskin and
ADSCs in vitro.
Fig. 4.
[93]Fig. 4
[94]Open in a new tab
The differentially expressed proteins (DEPs) among the MS+ADSC group,
the ADSC group, and the MS group. a Venn diagram showed there were 31
common DEPs detected in the MS+ADSC group compared to the ADSC group
and MS group. R Bioconductor package and golots were performed to
obtain heat map of DEPs in the MS+ADSC group compared with the ADSC
group (b) and MS group (c), with the cluster analysis results
GO/KEGG enrichment analysis of differentially expressed proteins
For GO enrichment analysis, the comparison of the MS+A group versus the
MS group and MS+A group versus a group showed similar results
(Fig. [95]5a, b). There were three notable cellular component (CC)
enrichment terms, including vesicle lumen, cytoplasmic vesicle lumen,
and secretory granule lumen. The biological process (BP) analysis
indicated that co-culture of microskin and ADSCs mainly had two
effects. One was positive regulation of cell migration and cellular
component movement, and the other was peptidyl-tyrosine
phosphorylation. The molecular function (MF) results showed that the
differentially expressed proteins in the MS+A group played critical
roles in enhancing the activity of cytokines, growth factors, receptor
regulator, and receptor ligand. Also, it made a difference in molecular
binding, including cytokine receptor binding, growth factor receptor
binding, and G protein-coupled receptor binding.
Fig. 5.
[96]Fig. 5
[97]Open in a new tab
GO/KEGG enrichment analysis of DEPs in the ADSC+MS group compared to
the ADSC group and MS group by DAVID online tool and R package
clusterProfiler. a GO enrichment results of the ADSC+MS group versus
ADSC group. b GO enrichment results of the ADSC+MS group versus MS
group. c KEGG pathway enrichment results of the ADSC+MS group versus
ADSC group. d KEGG pathway enrichment results of the ADSC+MS group
versus ADSC group. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes
and Genomes
The results of KEGG analysis of the MS+ADSC group versus the ADSC group
and the MS+ADSC group versus the MS group are highly similar
(Fig. [98]5c, d). According to the differentially expressed proteins,
KEGG analysis results indicated that PI3K-Akt [[99]22] and Jak-STAT
[[100]23] signaling pathways were involved with co-culture of microskin
and ADSCs in vitro, which may induce migration and proliferation of
fibroblasts and keratinocytes. Besides, PI3K-Akt [[101]24] and Ras
[[102]25] signaling pathways were also highly involved with co-culture
of microskin and ADSCs in vitro. As a result, the activity of these
signaling pathways may promote angiogenesis. The KEGG analysis
demonstrated that co-culture of microskin and ADSCs could activate
several signaling pathways which were highly related to enhance skin
wound healing through angiogenesis and cell migration and
proliferation.
PPI network analysis of the significant associated DEPs
To better analyze the protein-protein interaction in our study, a
section of common significant associated differentially expressed
proteins (FC > 1.5, p < .05) was chosen (Table [103]1). The PPI network
included 17 nodes and 57 edges (Fig. [104]6). In the PPI network, IL-6
(14), G-CSF (13), EGF (11), IP-10 (11), and ENA-78 (10) possessed a
large number of interactions (as displayed in the parentheses).
Moreover, these proteins might be the core protein in this PPI network
of a total of 17 nodes. Therefore, they were considered to be the
potential proteins in further studies of the treatment of microskin and
ADSCs to full-thickness skin defect.
Table 1.
The significant associated differentially expressed proteins in the
MS+ADSC group (FC > 1.5, p < .05)
Entrez ID Protein FC (MS+A versus MS) FC (MS+A versus ADSC)
3624 Activin A 4.34318 4.41328
58,191 CXCL16 2.24848 1.78707
1950 EGF 3.23495 11.55117
6374 ENA-78 6.73969 7.97758
1440 G-CSF 6.57958 11.01831
6346 I-309 7.55954 4.49978
3569 IL-6 8.25032 4.49978
3627 IP-10 6.27871 5.81403
3952 Leptin 2.56586 1.64033
3976 LIF 2.80270 2.75564
6355 MCP-2 4.34065 5.28009
6354 MCP-3 8.42093 4.27534
5228 PIGF 3.20651 3.85779
6352 RANTES 3.03501 2.01860
7043 TGFb3 10.52096 10.52096
7010 Tie-2 10.98094 10.98094
5329 uPAR 2.39436 1.82882
3791 VEGF R2 1.81428 10.66111
2324 VEGF R3 4.88643 11.08758
[105]Open in a new tab
Entrez, Entrez Gene ([106]http://www.ncbi.nlm.nih.gov/gene), FC fold
change, MS+A combination of microskin and ADSCs group, MS microskin
group, ADSC, ADSCs group
Fig. 6.
Fig. 6
[107]Open in a new tab
Intermolecular interactions of vital associated differentially
expressed proteins (DEPs). IL-6, G-CSF, EGF, IP-10, and ENA-78 held a
large number of interactions. The interaction relationship of DEPs was
shown as the lines. Network edges: line color represents the type of
interaction evidence from the interaction sources, line shape
represents the predicted mode of action, and line thickness represents
the strength of data support. Colored nodes: query proteins and first
shell of interactors; empty nodes: proteins of unknown 3D structure;
filled nodes: proteins of some known or predicted 3D structure
Discussion
For the reconstruction of a large area of wounded skin, there are
problems to be solved, including lack of microskin, wound contraction,
delayed vascularization, and scar formation [[108]26]. ADSCs were easy
to obtain and apply to many clinical trials and make a great result,
including skin regeneration, with the ability to regenerate wounded
tissue, enhancing vascularization, and inhibiting fibrosis [[109]18].
To our best knowledge, this is the first study of microskin and ADSCs
used in full-thickness skin defect mouse model. In the present study,
the mouse model showed that this combination of therapy could promote
full-thickness skin defects regenerating with faster wound closure and
more neovascularization. Our results indicated co-transplantation
microskin and ADSCs promote skin tissue regeneration by the secretions
of multiple cytokines which enhance pro-angiogenic.
Clinically, autograft microskin transplantation was one of the accepted
standard therapies to massive area skin damage [[110]2]. Microskin
transplantation had been employed to treatment for extensive skin
damage for years [[111]27]. However, there are still some limitations,
such as a limited abundance of donors, less revascularization, and
failure of transplantation [[112]28]. Evidence supported that ADSCs
could prolong the survival of skin grafts with angiogenic effect and
ameliorate microcirculatory alterations [[113]29, [114]30]. In our
work, the combination of microskin and ADSCs enhanced the repairing of
skin defect with a faster wound healing with less wound contraction,
compared to transplantation of ADSCs or microskin only. Interestingly,
the wound area at 14 days showed no statistical significance between
the MS group (7.039% ± 1.177%) and ADSC group (8.792% ± 0.743%). It
should be noted that the survival rate of microskin was increased in
the MS+ADSC group compared to the MS group (Additional file [115]5:
Figure S4B). This may reflect that the combination of microskin and
ADSCs could better accelerate skin wound healing.
To properly evaluate the therapeutic effect of the combination of
microskin and ADSCs, we performed an immunohistochemistry staining of
the wounded skin of each group. ADSCs, including their derived
extracellular microvesicles, were used to skin tissue regeneration
[[116]31]. It is confirmed that ADSC could facilitate angiogenesis in
tissue repair [[117]32]. In our study, we observed that the MS+ADSC
group showed more neovascularization by CD31 staining and less fibrosis
by α-SMA staining than other treatments. Interestingly, several skin
appendages were found in the MS+ADSC group, while few for the other
groups. All these data indicated that a better regeneration of skin
tissue happened in the MS+ADSC group. However, at day 14, the number of
neovascularization was not significant among the MS group
(11.40% ± 0.423%), ADSC group (10.30% ± 0.276%), and control group
(9.667% ± 0.27%) in this study. Treatment with ADSCs or microskin could
play a particular role at the early stage of angiogenesis during skin
wound healing. But the angiogenesis effect of ADSCs or microskin has no
remarkable superiority compared to the control group at the later stage
of skin wound healing. The reason might be the paracrine effect of
microskin, and ADSCs was weakened along with prolonging the time of
culture (Fig. [118]3c). We have noticed that there was still a signal
of neo-vascularization at 14 days post-injury. The VEGF staining of
wounded skin at day 14 post-injury showed the strong positive staining
was detected in the MS+ADSC group and ADSC group
(Additional file [119]6: Figure S5). In our vitro study, the expression
of VEGF in microskin culturing was declined after day 7, and this might
be the reason for the weaken VEGF staining of the wound sections at day
14 post-injury in the MS group. Normally, the number of vessels will
normalize and return to a level close to the normal skin at the late
stage of wound healing [[120]33]. In our study, the larger number of
neovascularization in the MS+ADSC group may due to the mutual promotion
of cytokine secretion, which was still unclear. The combined impact of
microskin and ADSCs showed a distinct advantage in angiogenesis which
accelerated concrescence of the skin wound.
We first wonder whether ADSCs could differentiate into epidermal cells
or endothelial cells to enhance wound healing in coaction with
microskin [[121]16, [122]17]. The co-culture system of microskin and
ADSCs showed that the keratin expression in ADSC was downregulated,
along with angiogenesis marker VWF and KDR. In other words, microskin
might not accelerate the differentiation ability of ADSCs in our study.
However, there were a few research reported the secretion function of
skin tissue culture [[123]34]. Therefore, we investigated the paracrine
effect of microskin. We observed that microskin could secret several
cytokines while cultured in the Transwell system, which was a
persistent secretion of EGF and VEGF. However, the secretion was
declining since day 7 of culture. The result of our in vivo experiment
also reflected that treatment of microskin or ADSCs only might not
access a satisfying outcome of angiogenesis.
The paracrine function was another critical function of ADSCs in wound
healing. Therefore, we further investigated the two-way paracrine
effects between microskin and ADSCs, and the data indicated that
combination of microskin and ADSCs could secrete various cytokines with
an up-expression, such as VEGF, IL-6, HGF, and EGF (Fig. [124]4b, c).
These cytokines have been confirmed that they play an essential role in
promoting angiogenesis [[125]35, [126]36]. Through GO enrichment
analysis, we found that the biological process of DEPs in the MS+ADSC
group was involved in cell migration, cellular component movement, and
peptidyl-tyrosine phosphorylation. The molecular function (MF) results
of them were involved in enhancing the activity of cytokines, growth
factors, receptor regulator, and receptor ligand. Moreover, three
important cellular component (CC) enrichment terms were detected,
including vesicle lumen, cytoplasmic vesicle lumen, and secretory
granule lumen. Also, the most significant pathways were enriched in
KEGG analysis, which are cytokine-cytokine receptor interaction and
PI3K-Akt and MAPK signaling pathways. These signaling pathways were
essential to wound repair [[127]22]. The paracrine of DEPs were
enriched in biological process and signaling pathway related to growth
factors and inflammation. The wound healing process after treatment of
microskin and ADSCs might involve the various cellular metabolic
process by the DEPs. By binding with other molecules (e.g., cytokines,
chemokines, and receptors) and activity of growth factors, receptors,
and ligands, they affect cell and cellular component movement,
inflammatory response, and phosphorylation. This combination of growth
factors and inflammatory factors stimulated wound healing-related cell
migration and angiogenesis for tissue regeneration.
Wound healing is a highly complex process and involves a variety of
complicated interactions among different resident cells extracellular,
extracellular matrix, soluble cytokines, and infiltrating leukocyte
subtypes. It has been confirmed that an appropriate inflammatory
response is necessary for healthy wound healing [[128]37]. However, the
excessive inflammatory response may lead to delay healing or
non-healing wounds [[129]38]. In this study, inflammatory factors
(e.g., IL-6) and growth factors (e.g., EGF) were upregulated in the
heat map and hold a larger number of interacting proteins in the PPI
network. IL-6 is considered as an inflammatory factor secreted from
cells that induce angiogenesis process in murine skin isografts
[[130]39]. With the close correlation of VEGF, treatment of epithelial
cell lines with IL-6 can induce the expression of VEGF mRNA to promote
angiogenesis [[131]40]. It is reported that IL-6 secreted from ADSCs
can stimulate angiogenesis, accelerate cutaneous wound healing, and
enhance recovery after ischemia/reperfusion (I/R) injury to increase
flap survival [[132]41–[133]43]. In the present study, we found
increasing neovascularization in the MS+ADSC group in mouse model and
an upregulation of IL-6 in protein array, which was consistent with
these previous studies. Our protein array detected a series of
upregulation of angiogenic factors, such as IL-6, Tie-2, uPAR, and
G-CSF. Besides, IL-6, G-CSF, EGF, IP-10, and ENA-78 might be the core
proteins in our study with a large number of interacting proteins. It
was probably that the combination of inflammatory factors and growth
factors might play a considerable part in angiogenesis.
Meanwhile, we have noticed that over a dozen highly relevant cytokines
(FC > 1.5, p < .05) were both detected in the MS+ADSC group in the
protein array, compared to the ADSC group and MS group (Table [134]1).
It was quite remarkable that a couple of angiogenic cytokines were
upregulated, including VEGFR2, G-CSF, Tie-2, and MCP-3. These cytokines
were confirmed highly related to angiogenesis by regulating the
migration, proliferation, and survival of vascular endothelial cells
and upregulating angiogenic cytokines [[135]44–[136]47]. We speculated
combination treatment of microskin and ADSCs promoted angiogenesis by
upregulating angiogenic cytokines.
Although the underlying mechanisms remain indistinct, our result
suggests that the cytokines, including VEGF, IL-6, HGF, and EGF, which
were upregulated in supernatants of co-culturing of microskin and
ADSCs, may play an essential role during the process of wound healing.
Furthermore, our data revealed the regulation of the two-way cytokine
between microskin and ADSCs. Most of the cytokines we detected were
up-expression in co-culturing of microskin and ADSCs. There should be a
positive feedback loop that upregulates the cytokines to promote wound
healing. However, future study is needed to find out the critical
cytokine and underlying signaling pathway in our study, as well as the
target cells. Therefore, we propose a possible mode of clinical
translation as follows: A combined transplantation of microskin and
ADSCs is operated to the wound site. The cytokines derived from
microskin and ADSCs could enhance the wound healing with a better
vascularization (Additional file [137]7: Figure S6).
There is still a limitation related to our study. Our investigation
focused on tissue-to-cell and cell-to-tissue interactions, while wound
healing including tissue-to-cell, cell-to-tissue, and cell-to-cell
interactions. Inflammation cells, extracellular matrix, and blood
supply also contributed to wound healing, not only adipose stem cells,
fibroblasts, and microskin. Further investigation is needed to figure
out which cytokine and signaling pathway are critical in vivo study.
Conclusions
Our present study demonstrated that autograft microskin combined with
adipose-derived stem cell could enhance the healing of a large-area
wound in a mouse model with better angiogenesis. The treatment with
microskin and ADSCs improve angiogenesis and reduce fibrosis with the
secretion of multiple cytokines. The interaction network of various
upregulated cytokines secreted by microskin and ADSCs, such as IL-6,
G-CSF, EGF, IP-10, and ENA-78, may play an essential role in promoting
wound healing. The combination of microskin and ADSCs may be a
promising therapy to enhance tissue regeneration of full-thickness skin
defect.
Additional files
[138]Additional file 1:^ (29.5KB, doc)
Table S1. Primers of quantitative reverse transcription–polymerase
chain reaction (qRT-PCR). (DOC 29 kb)
[139]Additional file 2:^ (4.5MB, tif)
Figure S1. Characterization and differentiation capacity of ADSCs. (A):
Flowcytometry analysis indicated that more than 98% of cultured cells
expressed CD73 (99.5%), CD90 (98.7%) and CD105 (98.8%), whereas a small
fraction of them expressed HLA-DR (1.7%), CD 45 (1.8%), CD 34 (1.9%),
CD 19 (0.1%) and CD 11b (0.2%). ISO-Alexa Flour, ISO-PE, and ISO-APC
were considered as controls (PE: phycoerythrin; APC: allophycocyanin)
(B): Oil Red O staining of ADSCs cultured in adipogenic media. Scale
bar = 50 μm (C): Alizarin red staining of ADSCs cultured in osteogenic
media. Scale bar = 50 μm (D): Alcian blue staining of ADSCs cultured in
chondrogenic media. Scale bar = 100μm. (TIF 4594 kb)
[140]Additional file 3:^ (2.1MB, tif)
Figure S2. Treatment of MS+ADSC obtained cosmetically appealing at day
14 post-injury compared to other groups. (A): MS+ADSC group (B): ADSC
group (C):MS group (D): control group. (TIF 2180 kb)
[141]Additional file 4:^ (6MB, tif)
Figure S3. Treatment of MS+ADSC reduced scar thickness at day 14
post-injury. (A): Representative photomicrographies of the scar tissue
which determined on H&E stained sections of wounded skin at day 14
post-injury (dashed lined area), and black lines indicated the scar
thickness. (B): Scar thickness in tissue sections of day 14 post-injury
skin. The data expressed are an average means ± SEM, n=5. ****, p
<.0001; **, p <.01; *, p <.05, compared to control group. (TIF 6116 kb)
[142]Additional file 5:^ (1.2MB, tif)
Figure S4. ADSCs play a role in improving the survival rate of micro
skin grafts. (A): The original area of micro skin grafts (rounded up by
yellow line) (B): The area of survival micro skin grafts (rounded up by
yellow line) (C): Representative of survival rate in MS+ADSC group and
MS group. The data expressed are an average means ± SEM, n = 5. ****, p
<.0001. (TIF 1249 kb)
[143]Additional file 6:^ (6.1MB, tif)
Figure S5. VEGF staining of wounded skin at day 7 and 14 post-injury.
There was a stronger positive staining of VEGF at wounded skin in
MS+ADSC group. Both day 7 and day 14 post-injury, the positive staining
was stronger in MS+ADSC group compared to other groups. (TIF 6239 kb)
[144]Additional file 7:^ (1.8MB, tif)
Figure S6. A possible mode of clinical translation about the combined
transplantation of micro skin and ADSCs. After a debridement of massive
injury and purchased autologous ADSCs, on the day of surgery, a
combined transplantation of micro skin and ADSCs is operated to the
wound site. The cytokines derived from micro skin and ADSCs could
enhance the wound healing with a better vascularization. (TIF 1798 kb)
Acknowledgments