Content uploaded by Zhaoyang Chen
Author content
All content in this area was uploaded by Zhaoyang Chen on Mar 06, 2021
Content may be subject to copyright.
Human decellularized adipose matrix derived hydrogel assists mesenchymal stem cells
delivery and accelerates chronic wound healing
Zhaoyang Chen1, 2
，
Bowen Zhang4, 5
，
Jun Shu2
，
Haiyang Wang3, 4
，
Yudi Han2
，
Quan Zeng3,
4
，
Youbai Chen2
，
Jiafei Xi3, 4
，
Ran Tao2
，
Xuetao Pei3, 4
，
Wen Yue3, 4,*
，
Yan Han2,*
1Medical School of Chinese PLA, Beijing, China
2Department of Plastic and Reconstructive Surgery, the First Medical Centre,
Chinese PLA General Hospital, Beijing, China
3Stem Cell and Regenerative Medicine Lab, Institute of Health Service and
Transfusion Medicine, AMMS, Beijing, China
4South China Research Center for Stem Cell & Regenerative Medicine, SCIB,
Guangzhou, China
5Beijing Institute of Radiation Medicine, AMMS, Beijing, China
Correspondence
Wen Yue, Stem Cell and Regenerative Medicine Lab, Institute of Health Service and
Transfusion Medicine, AMMS, No. 27 Taiping Road, Haidian District, Beijing 100850, China.
Email: yuewen0206@126.com
Yan Han, Department of Plastic and Reconstructive Surgery, the First Medical Centre, Chinese
PLA General Hospital, No. 28 Fuxing Road, Haidian District,
Beijing 100853, China.
Email: 13720086335@163.com
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jbm.a.37133
This article is protected by copyright. All rights reserved.
Abstract
Biological scaffolds based stem cell delivery methods have emerged as a promising approach for
tissue repair and regeneration. Here we developed a hydrogel biological scaffold from human
decellularized adipose matrix (hDAM) for human adipose-derived stem cells (hASCs) delivery to
accelerate chronic wound healing. The hDAM hydrogel was prepared by pepsin mediated
digestion and pH controlled neutralization. The morphology, survival, proliferation and
angiogenic paracrine activity of hASCs cultured in the hydrogel were assessed. Moreover, the
therapeutic efficacy of the hASCs-hydrogel composite for impaired wound healing was evaluated
by using a full-thickness wound model on diabetic mouse. The developed hDAM hydrogel was a
thermosensitive hydrogel, presented the biochemical complexity of native extracellular matrix
(ECM) and formed a porous nanofiber structure after gelation. The hydrogel can support hASCs
adhesion, survival and proliferation. Compared to standard culture condition, hASCs cultured in
the hydrogel exhibited enhanced paracrine activity with increased secretion of hepatocyte growth
factor (HGF). In the diabetic mice model with excisional full-thickness skin wounds, mice treated
with the hASCs-hydrogel composite displayed accelerated wound closure and increased
neovascularization. Our results suggested that the developed hDAM hydrogel can provide a
favorable microenvironment for hASCs with augmented regeneration potential to accelerate
chronic wound healing.
KEYWORDS
chronic wound healing, hydrogel, human adipose-derived stem cells, extracellular matrix,
mesenchymal stem cells
1. INTRODUCTION
Chronic wounds are skin defects that fail to restore structural and functional integrity in an
orderly and timely manner [1]. This kind of wounds is refractory and recurrent, which lead to
difficulties in clinical treatment and tremendous suffering in patients [2]. Recent studies have
shown that mesenchymal stem cells (MSCs) are effective for chronic wound healing. MSCs,
mostly derived from bone marrow, adipose tissue, umbilical cord, can promote wound closure and
angiogenesis through its regenerative paracrine effects and multi-lineage differentiation potential
[3]. However, the short retention time and low survival rate of transplanted stem cells limited the
therapeutic effects of MSCs, especially when using the traditional cell direct injection approaches
[4]. Thus, researchers have been making efforts to develop more effective cell delivery methods.
Biological scaffolds with cell delivery potentials are showing promising therapeutic effects in
chronic wound treatment [5]. These scaffolds provide a favorable microenvironment for cell
delivery and contribute to cell adhension, proliferation and differentiation [6]. With regard to
MSCs, previous studies have used sheet-formed biological scaffolds such as collagen sponge and
acellular dermal matrix to deliver MSCs [7, 8]. However, this kind of scaffold asked for additional
cell inoculation procedure and could not be well conformed to irregular wound geometry. Thus,
hydrogel biological scaffolds with sol-gel transition property are better options, which are
characterized by easy cell encapsulation and filling irregular injury site [9].
Hydrogels are highly hydrated polymer materials composed of synthetic or natural polymer
chains [10]. In contrast to synthetic hydrogels, natural derived hydrogels perform better on
measures of bioactive and biocompatible capacities. In addition, natural-derived hydrogels can
mimic the cell microenvironment in vivo, which make them ideal vehicles for cell delivery [11].
However, the most studied natural-derived hydrogels, like collagen or hyaluronic acid, only
contain individual component of the extracellular matrix (ECM), which compromised their
potentials in providing the complex matrix microenvironment necessary for stem cell function and
tissue regeneration [12]. Developing intact ECM-derived hydrogels from living tissues is
becoming a research focus in recent years.
In this study, we aimed to develop an ECM-derived hydrogel from human decellularized
adipose tissue matrix (hDAM) for delivering human adipose-derive stem cells (hASCs) to chronic
wounds. The hydrogel was prepared by pepsin mediated digestion and pH controlled
neutralization. The hydrogel is characterized in terms of biochemical composition, rheological
property and gel ultrastructure. The morphology, survival, proliferation and angiogenic paracrine
activity of hASCs cultured in the hydrogel were assessed. Moreover, the therapeutic efficacy of
the hASCs-hydrogel composite for impaired wound healing was evaluated using a full-thickness
wound model on diabetic mouse.
2 ∣ MATERIALS AND METHODS
2.1 ∣ Human adipose tissue collection
Fresh and sterile human subcutaneous adipose tissues were collected from female donors
(aged 18 - 35 years; BMI, <30; hemoglobin, >110 g/l) who had undergone abdominal or thigh
liposuction surgery at the First Medical Central of Chinese PLA General Hospital with informed
consent. Donors with diabetes mellitus, hypertension, serious systemic metabolic diseases or lipid
disorders were excluded. Patients were treated with tumescent anesthesia (tumescent solution:
1,000 ml of 0.9% normal saline + 10 ml of 2% lidocaine + 1 ml of 0.1% adrenaline). Fat was
suctioned using liposuction needles (3 mm in diameter) and under negative pressure via a 20 ml
syringe (used for hASCs isolation) or a liposuction machine (used for preparation of hDAM
hydrogel). After delivered to the lab on ice in sterile and airtight containers, the samples were
thoroughly rinsed with 0.9% sodium chloride solution to remove tumescent fluid, red blood cells
and cell debris. Samples obtained with the liposuction machine if not used for hDAM preparation
immediately would been placed in a -80 ℃ refri gerator for short-term storage. The research was
approved by the Ethics Committee of Chinese PLA General Hospital (No. S2017-059-10) and
conducted according to the 2000 Helsinki Declaration.
2.2 ∣ Preparation of hDAM hydrogel
hDAM was prepared from human adipose tissue with an enzyme and solvent-based
decellularization method as described previously [13]. Following decellularization, the extracted
hDAM was freezed at -80℃ overnight and then dried using Gamma 2-16 LSC Freeze Dryer
(Christ, Osterode, German) for 24 hours. Decellularization efficiency of the hDAM was evaluated
by residual dsDNA and triglyceride content assay. For residual dsDNA content assay, total DNA
was extracted from the hDAM with the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China)
and measured with the Quant-iT PicoGreen dsDNA Reagent and Kits (Invitrogen, Carlsbad, CA)
according to the manufacturer’s protocol (n=3) with the cutoff value of 50 ng/mg ECM (dry wt.).
The Cayman Triglyceride Colorimetric Assay kit (Chemical Inc, Ann Arbor, MI) was used to
measure the residual lipid content within the hDAM per the manufacturer’s instructions (n=3).
hDAM hydrogel was prepared by pepsin mediated digestion and pH controlled
neutralization. Briefly, 100 mg hDAM powder was digested with 10 mg 250 U/mg pepsin (Sigma,
St. Louis, MO) in 10 ml 0.01 M HCl at constant shaking for 72 hours at room temperature. The
hDAM pre-gel solution was formed by neutralizing the pH of hDAM digestion to 7.4 using 1 M
NaOH and 10 X alpha Minimum Essential Medium (α-MEM; Gibco, Waltham, MA), with
operating on ice. The hDAM hydrogel was prepared by incubating the pre-gel solution at 37 ℃ for
30 minutes. The hDAM pre-gel solution or hydrogel at 8 mg/ml was used for in vitro and in vivo
studies.
2.3 ∣ Biochemical composition analysis
The biochemical composition of the hDAM hydrogel was analyzed, including soluble
collagen content, sulfated Glycosaminoglycan (sGAG) content and protein profile.
hDAM digestion was diluted and assayed for soluble collagen content with the Sircol Soluble
Collagen Assay kit (Biocolor Ltd, Carrickfergus, United Kingdom) according to the
manufacturer’s instructions (n=3). Bovine collagen (500 μg/ml) was used as the reference standard
and 0.01 M HCl was used as the as the negative control. Absorbance at 555 nm of the reaction
solution was measured using spectramax M2 microplate reader (Molecular Devices, Sunnyvale,
CA).
hDAM pre-gel solution was assayed for sGAG content with the Blyscan sGAG Assay Kit
(Biocolor Ltd) according to the manufacturer’s instructions (n=3). Bovine tracheal chondroitin
4-sulfate (100 μg/ml) was used as the reference standard and ddH2O was used as the negative
control. Absorbance at 656 nm of the reaction solution was measured using the microplate reader.
The complex protein composition of the hDAM hydrogel was detected with Liquid
Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Representative sample of hDAM
digestion was ultra-centrifuged with Millipore 10 KD centrifugal filter unite (Billerica, MA, USA)
to remove undigested particles. The filtrates were collected, followed by desiccating, desalting and
desiccating. Dried sample was re-dissolved in 1% formic acid, and 1 μg of sample was used for
subsequent analysis. For LS-MS/MS analyzing, the sample was firstly separated by the Easy-nLC
1200 high performance liquid chromatography system (Thermo Scientific, Waltham, MA) at 200
nl/min, and then analyzed on Orbitrap Fusion Lumos mass spectroscopy (Thermo Scientific) at
data dependent acquisition mode (60,000 resolution (FWHM), m/z 375-1600, Higher Energy
Collision Dissociation (HCD) mode using 30% collision energy). Generated MS/MS spectra data
were transferred to peptide sequences by comparing to the Uniprot database using the in-house
Mascot server 2.3.2 (Matrix Science, London, UK). GO analysis was used to characterize the
cellular component, molecular function and biological process of the identified high score (>20)
peptides by searching the DAVID bioinformatics resources.
2.4 ∣ Rheological property measurement
The rheological property of the hDAM hydrogel was measured with Anton Paar MCR302
rheometer (Graz, Austrian) operating with a 40 mm parallel plate system. The temperature was
controlled within 0.01 ℃ using a Peltier plate. 1 ml of hD A M pre-gel solution placed on ice was
loaded onto the rheometer plate pre-cooled to 0 ℃. A f ter l oadi ng , the steady shear vi scosi ty w as
measured by applying a strain of 301% Pa at a frequency of 10 rad/s. The temperature was then
increased from 0 ℃ to 37 ℃ at 2 ℃ per 30 seconds to induce gelation, and a series of oscillatory
strain of 201% (0-29 ℃), 53% (31 ℃), 14% (33 ℃), 3% (35 ℃), and 1% (37 ℃) at a frequency of 10
rad/s were imposed to track the gelation kinetics.
2.5 ∣ Scanning electron microscopy
Scanning electron microscopy (SEM) was used to visualize the ultrastructure of the hDAM
hydrogel. For hydrogel samples preparation, gelation hDAM hydrogel were firstly fixed in cold
electron fixation solution (2.5% glutaraldehyde, Servicebio, Wuhan, China) for 24 hours at 4 ℃,
followed by secondary fixation in 1% osmic acid fix for 2 hours at room temperature. Fixed
samples were dehydrated in a graded series of alcohol (30, 50, 70, 90, 95, 100, 100% ethanol in
PBS) and isoamyl acetate for 15 minutes per wash. Samples were slowly dried in Quorum K850
critical point dryer (London, UK). After drying, the samples were sputter coated with gold for 30
seconds and imaged with Hitachi SU8100 SEM (Tokyo, Japan). A set of fiber network
characteristics such as fiber alignment, pore size, fiber diameter and node density (number of fiber
intersections per μm2) was measured from the acquired SEM images. The fiber architecture
features were automated extracted and quantified using the angiogenesis analyzer algorithm in
Image-J software (version 1.52a, NIH, USA).
2.6 ∣ Isolation and culture of hASCs
Primary hASCs were isolated from lipoaspirates according to the procedures described by Li
et al. [14]. Briefly, thoroughly rinsed adipose tissue was digested with equal volume 0.1%
collagenase I (Sigma) for 1 hour at 200 rpm and 37 ℃. A fter d i g esti on, the stromal vascul ar
fraction (SVF) pellets were collected by centrifuging the samples at 270 x g for 10 minutes at room
temperature. Collected cell pellets were resuspended with MSC serum-free medium (SANLY,
Beijing, China), plated to a 10 cm culture disk and cultured at 37 ℃ with 5% CO
2. The culture
medium was firstly changed 24 hours after plating to remove non-adherent cells, and then changed
every 2 days until the cells achieved 80-90% confluence. hASCs were passaged with TrypLE
Express (Gibco) to detach them from culture surface and cells at culture passage 3-6 were used in
the follow studies.
2.7 ∣ Multipotent differentiation potential test of hASCs
The multipotent differentiation potential of the cultured hASCs to adipocytes, osteoblasts and
chondrocytes were tested with the hASCs Differentiation Medium Kit (Cyagen, Guangzhou,
China) according to the manufacture’s protocol. hASCs cultured in Dulbecco’s modified Eagle
medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1%
penicillin-streptomycin (Gibco) were used as a control. After 3 weeks of adipogenic induction, the
lipid droplets formation capacity was evaluated by Oil-Red-O staining. After osteogenic
differentiation for 4 weeks, cells were fixed with 4% paraformaldehyde and stained with Alizarin
Red to visualize calcium deposition. Chondrogenic induction was conducted using a micromass
culture system. After 4 weeks of induction, cell aggregates were fixed with 10% formalin, paraffin
embedded, sectioned at 4 μm and stained with Alcian Blue to evaluate acid mucopolysaccharide
accumulation in ECM.
2.8 ∣ Flow Cytometry assay
The cultured hASCs were detached from plates, washed twice with PBS, and resuspended at
1.0×107 cells/ml. For fluorescent antibody labeling, 100 μl of sample was incubated with
monoclonal mouse anti-human antibodies for 30 minutes at room temperature. The anti-human
antibodies of PE-labeled CD29, PE-labeled CD34, PE-labeled CD105, PE-labeled CD117,
PE-labeled CD166, and PerCp-Cy5-labeled HLA-DR were purchased from BD Pharmingen (BD
Biosciences, San Diego, CA). The anti-human antibodies of APC-labeled CD31, APC-labeled
CD45 and FITC-labeled CD73 were purchased from eBioscience (San Diego, CA, USA).
Subsequently, the samples were washed twice with PBS, centrifuged for 5 minutes at 15294 x g,
and fixed with 4% paraformaldehyde. The labeled cells were detected on a Flow Cytometer (BD
FACSAria) and the data was analyzed using FlowJo software (version 10.0.7, BD Biosciences).
2.9 ∣ hASCs culture in the hDAM hydrogel
For assessing the biocompatibility of hASCs with the hDAM hydrogel, 2×106 hASCs were
suspended with 1 ml of hDAM pre-gel solution, and the MSC serum-free culture medium was
added after gelation.
hASCs morphology was observed with a microscopy and Hematoxylin and eosin (H&E)
staining after 7 days culture. For H&E staining, hASCs cultured in hDAM hydrogels were fixed
with 4% paraformaldehyde, paraffin embedded and sectioned at 4 μm.
hASCs viability was assessed with the LIVE/DEAD Viability/Cytotoxicity Kit (Molecular
Probes, Thermo Fisher Scientific) for 1, 3, 5 or 7 days with 150 μl/well of hASCs-hydrogel added
in triplicate to a 48-well plate. At each time point, the culture medium were aspirated, rinsed twice
with Dulbecco's Phosphate-Buffered Saline (DPBS) (Gibco). Then, 100 μl/well DPBS containing
2 μM Calcein-AM (excitation 494 nm, emission 517nm) and 5 μM Ethidium homodimer-1
(excitation 528 nm, emission 617nm) was added to each well, and incubated at 37 ℃ for 30
minutes. Stained cells were visualized with Nikon Eclipse Ti fluorescence microscopy (Tokyo,
Japan). The viability of hASCs was evaluated by quantification the ratio of viable cells to total
cells.
hASCs proliferative activity was evaluated with Ki-67 flow cytometry analysis after a 2 day
cultivate period α-MEM basic medium. hASCs cultured within the hydrogel were collected by
digesting with LiberraseTM TL (50 μg/ml, Roche, Merck) for 30 minutes at 37 ℃ and 5% CO
2. The
collected cells pellets were washed twice with PBS, and resuspended at 1.0×107 cells/ml. For
Ki-67 fluorescent antibody labeling, 100 μl of samples were fixed with 200 μl
Fixation/Permeabilization solution for 10 minutes at room temperature. After fixation, washed
twice with 1 ml 1X BD Perm/WashTM buffer, centrifuged for 5 minutes at 2000 g. Subsequently,
added PE-labeled mouse anti-Ki-67 (1:50 dilution,BD Biosciences) and incubated for 30 minutes
at room temperature, and then washed again. The labeled cells were detected on a Flow Cytometer
(BD FACSAria) and the data was analyzed using FlowJo software (version 10.0.7, BD
Biosciences).
2.10 ∣ Angiogenesis cytokine assay
The angiogenic paracrine of hASCs cultured in the hDAM hydrogel were assessed by
collecting the conditioned medium for antibody array assay (n=3). 1×106 hASCs/well suspended
in 1 ml of the MSC serum-free medium or 1 ml of the hDAM pre-gel solution were seeded on
6-well plates. After hASCs adherence or the hydrogel gelation, 3 ml of α-MEM medium was
added to replace the complete medium or 2 ml of α-MEM medium was added to cover the
hydrogel. After 48 hours cultivation, 1 ml/well of the supernatant was collected and centrifuged at
4 ℃, 425 x g for 10 minutes. For angiogenic cytokines assay, the collected medium were
concentrated 10 X times with a 10 KD centrifugal filter unite (Millipore) and the concentration of
cytokines were analyzed using the Quantibody Human Angiogenesis Array 1 (QAH-ANG-1;
RayBiotech, Norcross, GA) according to the manufacturer’s protocol (n=3). Briefly, completely
air dried glass slide was firstly blocked with 100 μl/well of sample diluent for 1 hour at room
temperature. After blocking, the diluent were decanted and 100 μl of standard cytokines or
samples were added to each well and incubated overnight at 4 ℃. The unconjugated proteins were
washed out using the wash buffer and deionized water. Subsequently, 80 μl of biotinylated
antibody cocktail was added to each well and incubated for 2 hour at room temperature, and then
washed again. To detect the conjugated antibody, 80 μl of Cy3 equivalent dye-conjugated
streptavidin was added to each well and incubated for 1 hour at room temperature, and then
washed again. The fluorescence was detected on InnoScan microarray scanner (Innopsys,
Carbonne, France), and the data was extracted and computed using the Q-Analyzer software. The
concentrations of factors in the conditioned media were reported after compensated for.
2.11 ∣ Animal study
All animal experiments were performed according to relevant institutional guidelines and
regulations of Beijing Medical Experimental Animal Care Commission and were approved by the
Animal Research Ethics Committee of Chinese PLA General Hospital (No. 2017-x13-25).
Twenty 9-week-old male KK/Upj-Ay/J mice (diabetic mice) were purchased from HFK
Biotechnology (Beijing, China) and randomized to four experiment groups: control, hydrogel
dressing, local hASCs injection, or hASCs-hydrogel composite treatment. After intraperitoneal
induction of anesthesia with 1% pentobarbital sodium, the hair of dorsum and abdomen were
shaved and depilated. Two 8 mm full-thickness wounds were created on either side of the dorsal
midline with a skin biopsy punch (Electron Microscopy Sciences, Hatfield, UK). For the hASCs
injection group, 2.4×105 hASCs suspended in 60 μl of α-MEM were injected subcutaneously
around the wound edge. For the hASCs-hydrogel composite or hydrogel dressing group, 60 μl of
hDAM hydrogel with/without 2.4×105 hASCs were transferred to the wounds after gelation. For
the control group, 60 μl of α-MEM were injected subcutaneously around the wound edge. All
wounds were covered with a transparent occlusive dressing (Smith&Nephew, Watford, UK).
Photographs were taken at days 0, 7, 10 and 14 after wound with a paper circle (inner
diameter 1.2 cm, outer diameter 1.6 cm) placed on the wound as a reference. Wound area was
measured using the Image-J software and the percentage of original wound area was calculated at
each time point. At the end of observation, wound samples were harvested for histological analysis
using paraffin sections. H&E staining was used to evaluate wound healing quality and
immunohistochemical (IHC) staining with antibody to CD31 (1:200 dilution; Servicebio) were
used to evaluate wound neovascularization. The dermal microvessel density (MVD) was counted
using four hpf at 200 X from each group by three independent blinded observers.
2.12 ∣ Statistical Analysis
All the experimental data are expressed as mean ± standard deviation. The student’s unpaired
t test was used for two groups comparison when the data meet normality and homogeneity.
ANOVA with an appropriate post-hoc test was used for multiple comparison. All the statistical
work were performed with SPSS 18.0 statistical software and p < .05 was considered as statistical
significance.
3 ∣ RESULTS
3.1 ∣ Preparation of hDAM hydrogel
The preparation process of hDAM hydrogel involves decellularization of adipose tissue,
freeze-drying, grinding, pepsin digestion and pH neutralization (Figure 1a). After 5 days of
decellularization, a small volume of white and hydrated hDAM was obtained. Feeze-dried hDAM
was a loose and porous material, and the average production per 100 ml of lipoaspirate was 142.4
± 43.4 mg (dry weight) (n=6). Fine powdered hDAM was obtained by cyomilling the lyophilized
hDAM with KZ-II grind mill (KangTao technology, Wuhan, China) and filtering through a 80
mesh sieve. Effectively decellularization and delipidization was confirmed, as compared to native
adipose tissue, the dsDNA content (49.59 ± 8.27 ng/mg vs. 4.74 ± 0.71 ng/mg, p < .001) and
triglyceride content (18.76 ± 1.02 μg/mg vs. 0.21 ± 0.16 μg/mg, p < .001) in lyophilized hDAM
were significantly reduced.
The hDAM hydrogel was successfully prepared by pepsin mediated solubilization and pH
controlled neutralization. After 72 hours pepsin digestion, the hDAM powder was solubilized to a
homogenous liquid. The pre-gel solution was found to be easily manipulated with a pipette.
Sol-gel transition property of the hDAM hydrogel was observed after incubating the pre-gel
solution at 37 ℃ for 30 mi nutes (Figure 1b). After gelation, the hD A M hydrogel can maintain a
stable form and stick to a well-plate cover (Figure 1c).
3.2 ∣ Characterization of hDAM hydrogel
To assess the potential of the hDAM hydrogel as a delivery vehicle for hASCs, we performed
the biochemical composition, rheological property, gel ultrastructure analysis.
The soluble collagen content in the hydrogel was 0.72 ± 0.04 mg/mg lyophilized hDAM, and
the sGAG content in the hydrogel was 2.34 ± 0.47 ug/mg lyophilized hDAM. There was total 142
proteins identified in the hDAM digestion by LS-MS/MS (Supplementary Table S1), and most of
them were various kinds of collagens and glycoproteins. 44 proteins with high score (>20)
(Supplementary Table S2) were included into the subsequently Gene ontology (GO) analysis
(Figure 2a). The cellular component ontology annotation results showed most of these proteins
were ECM origin. The molecular function ontology annotation results revealed these constitutes
mainly act as binding molecule. The biological process ontology annotation results indicated that
the top three processes in which these proteins participated were platelet degranulation, ECM
organization, and innate immune response.
The measurement results of rheological property displayed that the prepared hDAM hydrogel
was a thermosensitive hydrogel (Figure 2b). The temperature at which the storage modulus (G’)
equal to the loss modulus (G’’) is the sol-gel transition point and our experiments observed that
this transition point occurred at 31 ℃. W hen the temperature rose to 37 ℃, the G’ was roughl y 5
times the G’’. The viscosity of this hydrogel material was 91.05 mPa·s before gelation and showed
a substantial increase to 6207.7 mPa·s after gelation.
SEM images showed that the hDAM hydrogel possessed a loosely organized porous
nanofiber structure with interconnected pore and randomly oriented fibers (Figure 2c, left panel).
Visual inspection of the algorithm outputs showed that the fiber network features have been
accurately extracted (Figure 2c, right panel). Based on the measurement data, the average fiber
diameter of the hydrogel was 64.4 ± 10.7 nm, the average pore size was 0.112 ± 0.019 μm2, and the
average node density was 7.15 ± 0.53 nodes/μm2.
3.3 ∣ Isolation and identification of hASCs
The isolated and expanded hASCs were plastic-adherent in standard culture conditions and
exhibited typical fibroblast-like or spindle-shaped morphology (Figure 3a). Multipotent
differentiation test showed that the hASCs were able to differentiate into adipocytes, osteocytes,
and chondrocytes under standard in vitro differentiating conditions as demonstrated by Oil-Red-O,
Alizarin Red, and Alcian Blue staining (Figure 3b). Flow cytometry analysis results showed that ≥
98% of the cultured hASCs expressed the classical MSCs markers of CD29, CD73, CD90, CD105
and CD166, while these cells lacked expression (≤ 2.3% positive) of the endothelial cell specific
marker of CD31, the hematopoietic cells specific marker of CD45 and CD117, and the major
histocompatibility complex II (MHC-II) molecular of HLA-DR (Figure 3c).
3.4 ∣ hASCs survival and function in the hDAM hydrogel
To assess hASCs survival and function in the hDAM hydrogel, we evaluated the
morphology, viability, proliferation and angiogenic paracrine activity of hASCs cultured in the
hydrogel.
Microscopical and H&E staining images showed that hASCs cultured in the hydrogel
exhibited a healthy fibroblast-like phenotype (Figure 4a), indicating they were attached to the
hydrogel bioscaffold. The high viability of hASCs embedded in the hydrogel was similar to that in
standard condition over a period of seven days according to the LIVE/DEAD fluorescent staining
results (Figure 4b). The Ki-67 flow cytometric assay results showed hASCs within the hydrogel
proliferated significantly faster compared to those cultured on plastic surface (Hydrogel 34.3 ±
1.3 %; vs. Plated 20.3 ± 1.1 %, p<0.001) (Figure 4c).
Angiogenic cytokines assay was used to evaluate the paracrine activity of hASCs cultured in
the hDAM hydrogel (Figure 4d, e). The results showed that hASCs secreted a large amount of
hepatocyte growth factor (HGF), vascular growth factor-A (VEGF-A), angiopoietin (ANG) and
ANG-2. Compared to those cultured on plastic plate, hASCs cultured in the hDAM hydrogel
secreted a significantly high level of HGF (404.57 ± 30.23 pg/mL vs. 1182.54 ± 90.26 pg/mL, p <
.001) .
3.5 ∣ Effect of hASCs-hydrogel composite on impaired wound healing
We utilized the diabetic mice model with impaired wound healing ability, and created
excisional full-thickness wounds to test the therapeutic effect of hASCs-hydrogel composite in
vivo (Figure 4a). Wounds healed significantly faster when treated with hASCs-hydrogel
composite or local hASCs injection compared to those of control group (Figure 4b, c). Moreover,
there was a significant acceleration of wound closure at day 7 in hASCs-hydrogel composite
treated group (28.6 ± 5.2% original wound area, p = .002) compared to local hASCs injection
wounds (44.8 ± 6.9%). Wounds treated with hydrogel dressing showed improved healing only at
day 10, but no difference at day 7 or day 14 relative to those of control group. In addition to
accelerate wound closure, hASCs-hydrogel composite treatment also displayed an improved effect
on skin architecture regeneration, which included a better restoration of cutaneous appendages and
increase of dermis thickness (Figure 5d).
To assess the wound neovascularization, we performed CD31 IHC staining and MVD
assessment (Figure 5e, f). The MVD of wounds on healing Day 14 was significantly increased
when treated with hASCs-hydrogel composite compared to other three groups (68.7 ± 11.1 vessels
per high power fields (hpf); vs. hASCs injection, p = 0.02; vs. hydrogel dressing, p = 2.6×10-4; vs.
control, p = 1.3×10-6). Moreover, compared to untreated wounds, neovascularization was also
significantly augmented in wounds treated with local hASCs injection or hydrogel dressing (28.3
± 4.8 vessels per hpf; vs. hASCs injection, p = 2.6×10-4; vs. hydrogel dressing, p = 3.1×10-4).
However, there was no significant difference in MVD between wounds treated with local hASCs
injection and hydrogel dressing (52.0 ± 10.1 vs. 42.5 ± 5.2 vessels per hpf, p = .07).
4 ∣ DISCUSSION
In this study, we developed a hydrogel biological scaffold from hDAM for hASCs delivery to
accelerate chronic wound healing. The developed hDAM hydrogel was a thermosensitive
hydrogel, retained the biochemical complexity of the native ECM and formed a porous nanofiber
structure after gelation. The hydrogel can support hASCs adhesion, survival and proliferation，and
hASCs cultured in the hydrogel exhibited enhanced paracrine activity. In the diabetic mice model
with excisional full-thickness skin wounds, mice treated with the hASCs-hydrogel composite
displayed accelerated wound closure and increased neovascularization.
Previous studies have suggested that biological scaffolds based cell delivery methods were
beneficial for MSCs engraftment in chronic wound healing [15]. Our study demonstrated that
delivery of hASCs in hDAM hydrogel can accelerate impaired wound closure and promote
angiogenesis. Traditionally, biological scaffolds used by other studies were sheet formed like
collagen sponge or acellular dermal matrix, which usually requested additional inoculation and
incubation process for cell attachment before delivery to wound beds and could not adapt well to
the irregular defect geometry [7, 8]. The hDAM hydrogel we developed was a liquid solution
before gelation, making it easier to encapsulate cells. Moreover, its sol-gel transition property is
more conducive to cell delivery and site attachment. In contrast to synthetic polymeric hydrogels,
the hDAM hydrogel is derived from native ECM of adipose tissue, and therefore contains
structural and functional biomolecules secreted by resident cells, thus in favor of communicating
with stem cells in an interactive and dynamical manner. This kind of hydrogels can also act as an
inductive template for constructive remodeling in tissue repair and regeneration through
recruitment of endogenous stem and progenitor cells, antimicrobial activity, and modulation of
macrophage polarization [16].
The paracrine mechanism of MSCs in accelerating wound healing has been confirmed by
many studies [17]. Previous researchers have reported that tissue engineering approaches
enhanced the paracrine function of MSCs. However, these results were mainly obtained from
culturing of MSCs on electrospun fiber scaffolds [18, 19]. Our study demonstrated that the hDAM
hydrogel culture condition can also increase the angiogenic cytokines secretion of hASCs. The
enhanced secretome may account for augmented neovascularization in vivo, because increased
HGF secreted by hASCs is a trophic factor which can facilitate neovascularization during wound
healing [20]. The upregulated pro-angiogenic cytokines secretion of hASCs cultured in the hDAM
hydrogel may share a similar down-stream signal pathway with stimulated secretion of MSCs due
to hypoxia condition [21]. In-depth understanding of the interaction between materials and stem
cells can help guide the preparation of optimized materials to maximize the therapeutic efficacy of
stem cells. This finding suggests that the prepared hDAM hydrogel can provide a favorable
functional niche for MSCs and improve their regeneration potential.
The relatively long gelation time of the hDAM hydrogel after injection into wound area may
be one obstacle for its clinical application. But this could be improved by mixed with synthetic
materials or cross-linking agents [22]. Another thing need to be noted is that, since this material is
derived from human adipose tissue, attention should be paid to possible transmission of infectious
diseases and the difference in tissue activity between donors. However, this problem can be solved
by strict screening of infectious diseases and careful evaluation and selection of donors [23].
Despite the above mentioned shortcomings, the developed hDAM hydrogel is still a promising
bioscaffold material for chronic wounds. On one hand, human tissue-derived materials have
advantages over xenogeneic-derived materials in bioactivity, biocompatibility and
biodegradability [24]. On the other hand, if clinical application of stem cell transplantation in
wound treatment is restricted out of concern about ethical disputes, our hDAM hydrogel still has
potential as a carrier for exosomes or conditioned medium of stem cells [25, 26]. For example, van
Dongen et al have made a research on adipose tissue-derived ECM hydrogels as a release
platform for secreted paracrine factors, which provided a promising therapeutic modality to
promote several important wound healing-related processes by releasing factors in a controlled
way [27].
5 ∣ CONCLUSION
A ECM-derived hydrogel was developed from human decellularized adipose tissue by pepsin
mediated digestion and pH controlled neutralization. The developed hDAM hydrogel was a
thermosensitive hydrogel, presented the biochemical complexity of native ECM and formed a
porous nanofiber structure after gelation. The hydrogel can support hASCs adhesion, survival and
proliferation. Compared to standard culture condition, hASCs cultured in the hydrogel exhibited
enhanced paracrine activity with increased secretion of HGF. In the diabetic mice model with
excisional full-thickness skin wounds, mice treated with the hASCs-hydrogel composite displayed
accelerated wound closure and increased neovascularization. Our results suggested that the
developed hDAM hydrogel can provide a favorable microenvironment for hASCs with augmented
regeneration potential to accelerate chronic wound healing.
ACKNOWLEDGMENTS
This study was supported by the National Key R&D Program of China [2017YFA0103100,
2017YFA0103103, 2017YFA0103104]; and the Guangzhou Health Care and Cooperative
Innovation Major Project [201704020224, 201803040011, 201803040008, 201803040005].
DATA AVAILABILITY STATEMENT
All the data that support the findings of this study are availability in the supplement material of
this article.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
REFERENCES
1. Eming SA, Martin P, Tomic M. Wound repair and regeneration: mechanisms, signaling, and
translation, Sci Transl Med. 2014; 6: 265sr6.
2. Frykberg RG, Banks J. Challenges in the Treatment of Chronic Wounds, Adv Wound Care.
2015; 4: 560-582.
3. Hu MS, Borrelli MR, Lorenz HP, et al. Mesenchymal Stromal Cells and Cutaneous Wound
Healing: A Comprehensive Review of the Background, Role, and Therapeutic Potential, Stem
Cells Int. 2018; 2018: 6901983.
4. Lee DE, Ayoub N, Agrawl DK. Mesenchymal stem cells and cutaneous wound healing:
novel methods to increase cell delivery and therapeutic efficacy, Stem Cell Res Ther. 2016; 7.
5. Dash BC, Xu Z, Lin L, et al. Stem Cells and Engineered Scaffolds for Regenerative Wound
Healing, Bioengineering (Basel). 2018; 5: 23.
6. Turner NJ, Badylak SF. The Use of Biologic Scaffolds in the Treatment of Chronic
Nonhealing Wounds, Adv Wound Care. 2015; 4: 490-500.
7. O'Loughlin A, Kulkarni M, Creane M, et al. Topical administration of allogeneic
mesenchymal stromal cells seeded in a collagen scaffold augments wound healing and increases
angiogenesis in the diabetic rabbit ulcer, Diabetes. 2013; 62: 2588-2594.
8. Nie C, Zhang G, Yang D, et al. Targeted delivery of adipose-derived stem cells via acellular
dermal matrix enhances wound repair in diabetic rats, J Tissue Eng Regen Med. 2015; 9:
224-235.
9. Yu L, Ding J. Injectable hydrogels as unique biomedical materials, Chem Soc Rev. 2008; 37:
1473-1481.
10. Drury JL, Mooney DJ. Hydrogels for tissue engineering: scaffold design variables and
applications, Biomaterials. 2003; 24: 4337-4351.
11. Jabbar E. Hydrogels for Cell Delivery, Gels. 2018; 4.
12. Spang MT, Christman KL. Extracellular matrix hydrogel therapies: In vivo applications and
development, Acta Biomater. 2018; 68: 1-14.
13. Zhou ZQ, Chen Y, Chai M, et al. Adipose extracellular matrix promotes skin wound healing
by inducing the differentiation of adiposederived stem cells into fibroblasts, Int J Mol Med. 2019;
43: 890-900.
14. Li J, Curley JL, Floyd ZE, et al. Isolation of Human Adipose-Derived Stem Cells from
Lipoaspirates, Methods Mol Biol. 2018; 1773: 155-165.
15. Altman AM, Yan Y, Matthias N, et al. IFATS collection: Human adipose-derived stem cells
seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury
model, Stem Cells (Dayton, Ohio). 2009; 27: 250-258.
16. Saldin LT, Cramer MC, Velankar SS, et al. Extracellular matrix hydrogels from
decellularized tissues: Structure and function, Acta Biomater. 2017; 49: 1-15.
17. Lee SH, Jin SY, Song JS, et al. Paracrine effects of adipose-derived stem cells on
keratinocytes and dermal fibroblasts, Ann dermatol. 2012; 24: 136-143.
18. Carter K, Lee HJ, Na KS, et al. Characterizing the impact of 2D and 3D culture conditions
on the therapeutic effects of human mesenchymal stem cell secretome on corneal wound healing
in vitro and ex vivo, Acta Biomater. 2019; 99: 247-257.
19. Su N, Gao PL, Wang K, et al. Fibrous scaffolds potentiate the paracrine function of
mesenchymal stem cells: A new dimension in cell-material interaction, Biomaterials. 2017; 141:
74-85.
20. Ding S, Merkulova T, Han ZC, Tobelem, G. HGF receptor up-regulation contributes to the
angiogenic phenotype of human endothelial cells and promotes angiogenesis in vitro, Blood.
2003; 101: 4816-4822.
21. Muscari C, Giordano E, Bonaf F, et al. Priming adult stem cells by hypoxic pretreatments
for applications in regenerative medicine, J Biomed Sci. 2013; 20: 63.
22. Zhu J, Marchant RE. Design properties of hydrogel tissue-engineering scaffolds, Expert
Rev Med Devices. 2011; 8: 607-626.
23. Chen FM, Liu X. Advancing biomaterials of human origin for tissue engineering, Prog
Polym Sci. 2016; 53: 86-168.
24. Badylak SF, Freytes DO, Gilbert TW. Extracellular matrix as a biological scaffold material:
Structure and function, Acta Biomater. 2009; 5: 1-13.
25. Wu P, Zhang B, Shi H, et al. MSC-exosome: A novel cell-free therapy for cutaneous
regeneration, Cytotherapy. 2018; 20: 291-301.
26. Zhou P, Li X, Zhang B, et al. A human umbilical cord mesenchymal stem cell-conditioned
medium/chitosan/collagen/β-Glycerophosphate thermosensitive hydrogel promotes burn injury
healing in mice, Biomed Res Int. 2019; 2019: 5768285.
27. Van Dongen JA, Getova A, Brrouwer LA, et al. Adipose tissue-derived extracellular matrix
hydrogels as a release platform for secreted paracrine factors. J Tissue Eng Regen Med. 2019; 13:
973-985.
FIGURE LEGENDS
FIGURE 1 Preparation of hDAM hydrogel from human adipose tissue. Major steps of the
hDAM hydrogel preparation (a). Sol-gel transition property of the hDAM hydrogel (b).
Macroscopic appearance of the hDAM hydrogel (c).
FIGURE 2 Characterization of hDAM hydrogel. Gene ontology (GO) analysis results of the
high score proteins (>20) identified by LC-MS/MS (a). Rheological property of the hDAM
hydrogel (b). Scanning electron micrographs of the hDAM hydrogel (left panel) and the extracted
fiber network characteristics by algorithm (right panel) (c).
FIGURE 3 Identification of hASCs. Morphology of cultured hASCs (a). Multipotent
differentiation of hASCs (b), adipogenic differention was evaluated by Oil-Red-O staining of lipid
droplets (first column), osteogenic differentiation was evaluated by Alizarin Red staining of
deposited calcium (second column), chondrogenic differentiation was evaluated by Alcian Blue
staining of accumulated acid mucopolysaccharide in extracellular matrix (third column), scale bar
= 50 μm. Surface markers of hASCs as detected by flow cytometry (c).
FIGURE 4 hASCs survival and function in the hDAM hydrogel. Microscopy image and H&E
staining of hASCs cultured in the hydrogel for 7 days (a), scale bar = 100 μm. Live/Dead assay of
hASCs viability in the hydrogel (b), right panel is a representative fluorescence image at 7 days,
scale bar = 100 μm. Ki-67 flow cytometric analysis of hASCs proliferation in the hydrogel (c).
Heatmap of angiogenic cytokines concentration in conditioned medium from hASCs cultured in
the hydrogel and plastic plate (d), the scaled values represent the concentration of proteins
(pg/mL), and relative amounts of proteins in the conditioned medium from hASCs cultured in the
hydrogel compared to plastic plate (e; *p < .05, **p < .01, ***p < .001).
FIGURE 5 Effect of hASCs-hydrogel composite on impaired wound healing. Excisional
full-thickness wound model preparation and hASCs-hydrogel composite engraftment (a). Gross
photos (b) and Percentage of original wound area (c). H&E staining of wounds at day 14 (d), scale
bar = 100 μm. CD31 IHC staining of wounds at day14 (e), scale bar = 200 μm. and Quantification
of MVD (f; *p < .05, **p < .01, ***p < .001).
FIGURE 1
FIGURE 2
Accepted Article
This article is protected by copyright. All rights reserved.
FIGURE 3
Accepted Article
This article is protected by copyright. All rights reserved.
FIGURE 4
Accepted Article
This article is protected by copyright. All rights reserved.
FIGURE 5
Accepted Article
This article is protected by copyright. All rights reserved.
