Acta Biomaterialia 92 (2019) 160–171

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Full length article

A collagen scaffold loaded with human umbilical cord-derived

mesenchymal stem cells facilitates endometrial regeneration and
restores fertility
Liaobing Xin a,b,c,1, Xiaona Lin a,c,1, Yibin Pan a,c, Xiaowen Zheng b, Libing Shi a,c, Yanling Zhang a,c, Lie Ma b,c,⇑,
Changyou Gao b, Songying Zhang a,c,⇑
a
Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China
b
MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
c
Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, No. 3 Qingchun East Road, Jianggan District, Hangzhou 310016, China

a r t i c l e i n f o a b s t r a c t

Article history: In women of reproductive age, severe injuries to the endometrium are often accompanied by endometrial
Received 22 November 2018 scar formation or intrauterine adhesions (IUAs), which can result in infertility or miscarriage. Although
Received in revised form 17 April 2019 many approaches have been used to treat severe IUAs, high recurrence rates and endometrial thinning
Accepted 6 May 2019
have limited therapeutic efficiency. In this study, a collagen scaffold (CS) loaded with human umbilical
Available online 7 May 2019
cord-derived mesenchymal stem cells (UC-MSCs) was fabricated and applied for endometrial regenera-
tion. The CS/UC-MSCs promoted human endometrial stromal cell proliferation and inhibited apoptosis
Keywords:
in vitro through paracrine effects. In a model of endometrial damage, transplantation with the CS/UC-
Collagen scaffold
Umbilical cord-derived mesenchymal stem
MSCs maintained normal luminal structure, promoted endometrial regeneration and collagen remodel-
cells ing, induced intrinsic endometrial cell proliferation and epithelium recovery, and enhanced the expres-
Endometrial regeneration sion of estrogen receptor a and progesterone receptor. An improved ability of the regenerated
Intrauterine adhesion endometrium to receive embryos was confirmed. Together, our results indicate that the CS/UC-MSCs pro-
Fertility restoration moted endometrial structural reconstruction and functional recovery. Topical administration of the CS/
UC-MSCs after trans-cervical resection of adhesions might prevent re-adhesion, promote endometrium
regeneration and improve pregnancy outcomes for patients with severe IUAs.

Statement of Significance

Intrauterine adhesions due to severe endometrium injuries happen frequently in clinic and become one
of the crucial reasons for women’s infertility or miscarriage. Therefore, how to regenerate the damaged
endometrium is a big challenge. In this study, a collagen scaffold (CS) loaded with human umbilical
cord-derived mesenchymal stem cells (UC-MSCs) was fabricated and applied for endometrium regener-
ation. Herein, UC-MSCs, known for low immunogenicity and high proliferative potential, exhibit promis-
ing potential for endometrium regeneration; and collagen scaffolds provide suitable physical support. It
was proved that transplantation with CS/UC-MSCs promoted endometrial regeneration and fertility
restoration. It suggested that topical administration of CS/UC-MSCs in uterus could be a promising strat-
egy for patients suffering severe intrauterine adhesion and infertility.
Ó 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Human endometrium is a dynamic and regenerative tissue

⇑ Corresponding authors at: Key Laboratory of Reproductive Dysfunction Man- composed of epithelial cells, stromal cells, vascular smooth cells,
agement of Zhejiang Province, No. 3 Qingchun East Road, Jianggan District, and vascular endothelial cells. The endometrium can be struc-
Hangzhou 310016, China. turally divided into two zones: the upper functional layer and
E-mail addresses: liema@zju.edu.cn (L. Ma), zhangsongying@zju.edu.cn
lower basal layer. During a menstrual cycle, the functional layer
(S. Zhang).
1
These authors contributed equally.
is shed while the permanent basal layer regenerates a brand-new

https://doi.org/10.1016/j.actbio.2019.05.012
1742-7061/Ó 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
 L. Xin et al. / Acta Biomaterialia 92 (2019) 160–171 161

functional layer according to fluctuating levels of estrogen and pro- articular cartilage [29], periodontal tissue [30], and brain [31]. In
gesterone. However, intrauterine adhesions (IUAs), which result the reproductive field, most studies have focused on regeneration
from repeated curettage, infections and intrauterine operations, of the uterine wall or even the entire uterus. For example, an engi-
can cause damage to the endometrial basal layer [1,2]. Subsequent neered uterine tissue was constructed by encapsulating human
failure of re-epithelization, inflammation and fibrotic tissue depo- endometrial stromal cells, epithelial cells and smooth muscle cells
sition lead to shrinking of the uterine cavity and endometrium, into a collagen/Matrigel scaffold in vitro [32]. In addition, the uter-
inactive glands and poorly vascularized stroma. A meta-analysis ine horns of rats were reconstructed by the transplantation of stem
showed that approximately 19% of women experiencing miscar- cell-loaded CS grafts after partial excisions [33–35]. However,
riages were diagnosed IUAs within 12 months. Among these regeneration of an entire uterus is a major challenge because of
women, 58.1%, 28.2% and 13.7% had mild, moderate and severe the complexity of uterine tissue. Moreover, the primary cause of
IUAs, respectively [1]. Importantly, severe IUAs may be associated IUA is not disorder of the uterus or uterine walls, but damage to
with infertility, including menstrual disorders, spontaneous abor- the endometrium. In this study, we aimed to develop a CS loaded
tion and pregnancy complications [3,4]. Many strategies, including with UC-MSCs for endometrial regeneration. We demonstrated
hysteroscopy, hormone therapy, intrauterine devices and antibi- that the CS/UC-MSCs promoted endometrial stromal cells prolifer-
otics, have been used for treatment of IUAs [5–7]. However, ation and inhibited apoptosis in vitro via paracrine effects. The effi-
endometrial regeneration remains a significant challenge. Current cacies of the CS/UC-MSCs in restoring the structure and function of
treatments can temporarily normalize the shape and volume of the endometrium were assessed in a rat model of endometrial
the uterine cavity but cannot restore the structure and function damage. Finally, regenerative outcomes were assessed using a fer-
of the endometrium, leading to IUA reformation. The recurrence tility test.
rate of severe IUAs after hysteroscopy is up to 62.5% [3,8], and
the pregnancy rate following surgical treatment is only 33.3% [9].
2. Materials and methods
Multiple approaches have been proposed to prevent recurrence
of IUAs after surgery, such as application of hyaluronic acid gel in
2.1. UC-MSC monoculture, flow cytometric analysis and multipotent
humans [10], transplantation of freeze-dried amnion grafts in
differentiation analysis
humans [11], or transplantation of oral mucosal epithelial cell
sheets in rats [12]. These interventions can prevent the recurrence
Frozen UC-MSCs at passage 3 were purchased from Boyalife
of IUAs to some degree, but do not restore a functional endome-
Group Ltd. (China). After thawing, the cells were seeded in 10-cm
trium. Therefore, the question of how to reconstruct an endome-
culture dishes (1
106 cells/dish) in Dulbecco’s modified Eagle’s
trium with normal morphology and function is a vital issue for
medium/F12 (DMEM/F12, Gino Biological, Hangzhou, China) sup-
IUA treatment.
plemented with 10% (v/v) fetal bovine serum (FBS, SA112.02, Cell-
Several medications such as aspirin [13–15] and granulocyte
max, Beijing, China), 100 U/mL penicillin (Gibco), and 100 mg/mL
colony stimulating factor [16] are recommended to promote
streptomycin (Gibco). When UC-MSCs reached 80–100% conflu-
human endometrial regeneration by increasing blood perfusion.
ence, cells were subcultured in quadruplicate. UC-MSCs harvested
However, due to damaged revascularization and ischemia in severe
at passage 4–6 were used in this study.
IUA lesions, the clinical effects of these medications are limited
The phenotype and multipotent differentiation of UC-MSCs
[17].
were confirmed (Fig. S1). For flow cytometric analysis, UC-MSCs
It has become evident that endometrial stem/progenitor cells in
(1
106) were incubated with 1 mg each of fluorescein
both the functional and basal layers are responsible for endometrial
isothiocynate(FITC)-conjugated anti-human CD73 (Biolegend,
regeneration [18–20]. In IUAs, the numbers and function of endome-
344016, USA), phycoerythrin (PE)-conjugated anti-human HLA-
trial stem/progenitor cells are diminished due to the lack of func-
DR (Beckman, IM1639, USA), anti-human CD34 (Beckman,
tional endometrium [3]. Recently, cell therapy toward uterus have
A07776, USA), anti-human CD45 (Beckman, A07783, USA), anti-
been popular in treating endometrial dysfunction [20]. Mesenchy-
human CD19 (Beckman, A07769, USA), anti-human CD11b (Beck-
mal stem cells (MSCs) have emerged as a promising cell type for tis-
man, IM2581U, USA), PE/CF594-conjugated anti-human CD90
sue regeneration since they are characterized by multipotent
(Beckman, IM3703, USA), and PE/Cy7-conjugated anti-human
differentiation, high expansive potential, and immunomodulatory
CD105 (Biolegend, 323218, USA) antibodies for 30 min in the dark.
capability. Depending on the cellular source, MSCs can be classified
After washing twice with phosphate-buffered saline (PBS), the
as bone marrow-derived, umbilical cord-derived, adipose-derived,
samples were analyzed using a flow cytometer (Beckman Coulter,
and human menstrual blood-derived MSCs. Umbilical cord-
Fullerton, CA, USA). For osteogenic differentiation analysis, UC-
derived MSCs (UC-MSCs) have been regarded as a promising source
MSCs (5
103 cells/cm2) were treated with 0.1 lM dexametha-
for cell-based therapies because of their easy collection, low
sone (Sigma-Aldrich, St Louis, MO, USA), 10 mM b-glycerol phos-
immunogenicity, and high proliferative potential [21]. Applications
phate (Sigma-Aldrich, St Louis, MO, USA) and 50 lg/mL ascorbic
of UC-MSCs include bone defects [22], renal diseases [23], and
acid (Sigma-Aldrich, St Louis, MO, USA). After incubating for
peripheral nerve injuries [24]. However, few studies have reported
15 days, positive induction was observed by Alizarin Red staining.
the application of UC-MSCs to promote endometrial repair.
For analysis of adipogenic and chondroblast differentiation, cells
The main obstacle to treatment with MSCs is their low local
were incubated in adipogenic differentiation medium (Biological
persistence and utilization rate in the endometrium [25]. Only
Industries, Israel) or chondrogenic differentiation medium (Biolog-
0.59% or 0.65% of engrafted cells were observed around small
ical Industries, Israel) for 15 days according to the manufacturer’s
endometrial vessels following intrauterine infusion or tail vein
instructions. Positive induction was observed by oil red staining
injection in a murine model [26]. As critical components of tissue
and Alcian Blue staining.
engineering materials, porous scaffolds play an essential role in tis-
sue repair and regeneration. Collagen, a natural biomaterial and
the main component of the extracellular matrix, has been widely 2.2. Human endometrial stromal cell (HESC) isolation, culture and
utilized for tissue engineering scaffolds. A collagen scaffold (CS) immunofluorescence analysis
can provide a suitable physical support and microenvironment
for transplanted stem cells [27,28]. CSs loaded with stem cells have HESCs were obtained from fresh endometrial specimens as pre-
been applied for regeneration of many tissues and organs including viously described [36]. Informed consent was obtained from all
162 L. Xin et al. / Acta Biomaterialia 92 (2019) 160–171
patients prior to endometrium tissue collection. The procedures
were approved by the ethics committee of Sir Run Run Shaw
Hospital, Zhejiang University. In brief, endometrial specimens were
collected by biopsy from women with normal menstrual cycles and
rinsed with Hank’s Balanced Salt Solution (HBSS, Invitrogen, USA)
on ice. The samples were minced to <1 mm3 and incubated in
DMEM/F12 containing 0.25% (w/v) collagenase at 37 °C for
90 min. After digestion, stromal cells were obtained by passage
through a 40-mm nylon cell strainer (BD Biosciences) and washed
twice with HBSS. The stromal cells were seeded in 35-mm plates
(1.5
105 cells/well) in DMEM/F12 supplemented with 10% FBS,
100 U/mL penicillin (Gibco) and 100 lg/mL streptomycin (Gibco).
The cells were passaged until near confluence and cells were col-
lected at passage 4–6 for subsequent experiments.
HESC phenotypes were confirmed by immunofluorescence
analysis (Fig. S1C). Cells were fixed in 4% (v/v) paraformaldehyde
for 10 min, permeabilized with 0.2% (v/v) Triton X-100 for
10 min, blocked with 2% (w/v) bovine serum albumin (BSA, Gibco)
for 1 h and stained with rabbit anti-vimentin (1:100, ab92547,
Abcam, Cambridge, UK) or mouse anti-cytokeratin (1:100,
ab215838, Abcam, Cambridge, UK) antibodies diluted in 2% BSA
overnight at 4 °C. Sections were incubated with Alexa Fluor 488-
conjugated goat anti-rabbit IgG (1:300, ab150077, Abcam, Cam-
bridge, UK) and DyLight 549-conjugated goat anti-mouse IgG
(1:200, GAM5492, MultiSciences, Hangzhou, China) diluted in
PBS for 1 h at room temperature. Nuclei were stained with 40 , 6-
diamidino-2-phenylindole (DAPI) (20 lg/mL, Roche, Germany)
and observed using a fluorescence microscope (Leica, Germany).
2.3. Fabrication of CS/UC-MSCs
Collagen type I was obtained from bovine tendon by tryptic
digestion and acid dissolution as previously described [37]. The CS
was fabricated as previously described [38,39]. Briefly, the collagen
solution (0.5% w/v) was injected into a polytetrafluoroethylene
mold, frozen at 20 °C for 12 h and then lyophilized (Biocool, China)
for 24 h to obtain CSs. The CSs were treated by thermal dehydration
crosslinking at 105 °C under vacuum (Jinghong, China) for 15 h. To
fabricate the CS/UC-MSCs, 50 lL of UC-MSCs (1
107 cells/mL) were
placed onto a sterilized CS segment (2.5 cm
0.5 cm) and further
cultured in DMEM/F12-10% FBS for 3 h.
2.4. Characterization of CS/UC-MSCs
The CS/UC-MSCs were fixed with 4% paraformaldehyde for 24 h,
dehydrated with graded ethanol, embedded in paraffin and sec-
tioned at 5 lm thickness. Sections were stained with hematoxylin
and eosin (HE). The distribution of UC-MSCs within CSs after 12 h
culture was observed under an optical microscope (Nikon Eclipse
80i, Japan). The microstructures of CSs and UC-MSC morphology
within the scaffolds after 12 h were observed using scanning elec-
tron microscopy (SEM, Hitachi Model S-520, Japan). The SEM spec-
imens were fixed with 2.5% (v/v) glutaraldehyde for 24 h at 4 °C,
post-fixed with 1% osmium tetroxide, dehydrated in graded etha-
nol, dried under a critical point drier, and coated with gold. The
proliferative behavior of UC-MSCs in CSs and tissue culture
polystyrenes (TCPSs) was evaluated using a cell counting kit-8
(CCK-8) assay. Three samples from each group at different time
intervals were assessed in the CCK-8 assay (n = 3).
2.5. Effect of CS/UC-MSCs on HESC proliferation and apoptosis in vitro
A transwell co-culture system (Corning, NY) was used in this
study. For the cell proliferation assay, HESCs (passage < 6) were
seeded in a 24 well plate (1
105 cells/well) containing DMEM/
F12 and cultured for 2 h. A 0.4-lm pore insert containing a CS/
UC-MSCs (a 0.5
0.5 cm CS seeded with 2
105 UC-MSCs) was
placed on top of the HESC-seeded plate. HESCs cultured in
DMEM/F12 containing a blank insert and HESCs cultured in
DMEM/F12 containing 10% FBS were used as negative and positive
controls, respectively. The medium was refreshed every 24 h. A
CCK-8 assay (Dojindo, Shanghai, China) was used to evaluate the
proliferation of HESCs according to the manufacturer’s protocol.
Three samples from each group were assessed in the CCK-8 assay
(n = 3).
For cell apoptosis assays, HESCs were seeded into 6 well plates
and grown to 80% confluence. An insert containing a CS/UC-MSCs
(a 2
2 cm collagen scaffold seeded with 1
10 6 UC-MSCs) was
placed in each well using a procedure similar to the cell prolifera-
tion assay. A PE Annexin V Apoptosis Detection Kit I (559763, BD
Biosciences, USA) was used to measure HESC apoptosis after 48 h
co-culture according to the manufacturer’s instructions. In brief,
100 mL of HESCs (1
106 cells/mL) were incubated with 5 mL of
phycoerythrin and 5 mL of 7-amino-actinomycin (7-AAD) for
15 min in the dark. Three samples from each group were evaluated
in this assay (n = 3) and analyzed using a flow cytometer (BD
LSRFortessa TM Cell Analyzer, USA).
2.6. Enzyme-linked immunosorbent assay (ELISA)
Vascular endothelial growth factor (VEGF-A), transforming
growth factor (TGF-b1), and platelet-derived growth factor
(PDGF-BB) concentrations in the co-culture system were measured
by ELISA. The same trans-well co-culture system was used as for
the cell proliferation assay and culture medium was collected after
24 h co-culture. The medium was centrifuged at 800 rpm for 5 min.
The supernatant was collected and analyzed using a VEGF-A ELISA
Kit (Elabscience, E-EL-H0111c, Wuhan, China), a TGF-b1 ELISA Kit
(Elabscience, E-EL-H0110c, Wuhan, China) and a PDGF-BB ELISA
Kit (Elabscience, E-EL-H1577c, Wuhan, China) according to the
manufacturer’s protocols. Concentrations of VEGF-A, TGF- b1, and
PDGF-BB were quantified and expressed in pg/mL. Four samples
from each group were assessed in this assay (n = 4).
2.7. Rat model of endometrial damage and transplantation of CS/UC-
MSCs
Animal experiments were approved by the committee for ani-
mal ethics, Zhejiang University, and were monitored by the ethics
committee of Sir Run Run Shaw Hospital, Zhejiang University.
Female Sprague-Dawley (SD) rats (200–230 g, 7–9 weeks) were
purchased from the Experimental Animal Center of Zhejiang Pro-
vince (China) and housed in a controlled environment at 22 °C with
a 12 h/12 h light/dark cycle. The animals were allowed to acclima-
tize for 1 week prior to the experiments.
A rat model of endometrial damage was established as
described previously [12]. Briefly, all rats were anesthetized by
intraperitoneal injection of 2% sodium pentobarbital
(0.3 mL/100 g), and the uterine horns were exposed via an abdom-
inal incision. A 3-cm longitudinal incision was made in the uterus
to expose the inner endometrium. The endometrium was scraped
using a T10 scalpel blade until the surface of the uterus was rough
and bleeding. After washing with PBS (pH 7.4), a CS/UC-MSCs
(2.5 cm
0.5 cm) was transplanted onto the damaged endometrial
surface using a plastic device as previous described [12]. The
uterus was stitched using 6–0 absorbable sutures.
2.8. In vivo tracing of implanted CS/UC-MSCs
To trace implanted CS/UC-MSCs, 18 rats (36 uterine horns)
were used. At various time intervals (1, 3, 5, 7, 15 and 30 days
post-transplantation), 3 rats (6 uterine horns) were sacrificed and
 L. Xin et al. / Acta Biomaterialia 92 (2019) 160–171
uteri were harvested (n = 6). For HE staining, the uteri were fixed
with 4% paraformaldehyde overnight and sectioned at 5-lm thick-
ness. For immunofluorescence tracing of UC-MSCs labeled with
CM-Dil (C7000, Molecular Probes, Invitrogen, Eugene, USA)
[33,35,40,41], the samples were frozen in liquid nitrogen for
cryo-sectioning (Leica, Germany). Cell nuclei were stained with
DAPI (20 lg/mL, Roche, Germany), and observed using a fluores-
cence microscope (Leica, Germany).
2.9. Histological and immunohistological analysis
72 rats (144 uterine horns) were assigned randomly to four
groups: (i) the sham group (sham, n = 18 uterine horns), (ii) the
natural repair group (NR, n = 36 uterine horns), (iii) the CS group
(CS, n = 36 uterine horns), and (iv) the CS/UC-MSCs group (CS/
UC-MSCs, n = 54 uterine horns). Rats with damaged uteri trans-
planted with a CS/UC-MSCs were referred to as the CS/UC-MSCs
group. Rats transplanted with a CS or receiving no treatment were
referred to as the CS group or the NR group, respectively. Rats with
intact uteri without excision after abdominal incision were
referred to as the sham group. Samples with a length of 0.5 cm
were harvested from uterine wound sites near the cervix at each
time interval. The sections were stained with HE and Masson tri-
chrome using conventional protocols. The thickness of the regener-
ated endometrium was measured as the maximum vertical
distance from the luminal epithelium to the outer smooth muscle
including the uterine cavity or half of the maximum linear distance
between the smooth muscle excluding the uterine cavity under a
magnification of 40
. For immunohistochemistry, sections were
deparaffinized and rinsed with water. Antigen retrieval was per-
formed in 10 mM sodium citrate using a microwave. The slides
were washed in PBS containing 0.1% Tween 20, blocked in 5%
BSA for 1 h at room temperature, and incubated with primary anti-
bodies diluted in 5% BSA overnight at 4 °C. The primary antibodies
used were anti-Ki67 (1:200, ab15580, Abcam, Cambridge, UK),
anti-pan-cytokeratin (1:150, ab215838, Abcam, Cambridge, UK),
anti-estrogen receptor a (ERa) (1:200, ab79413, Abcam, Cam-
bridge, UK), anti-progesterone receptor (PR) (1:100, ab2765,
Abcam, Cambridge, UK), anti-VEGF-A (1:200, ab46154, Abcam,
Cambridge, UK), anti-TGFb1 (1:200, ab125287, Abcam, Cambridge,
UK), anti-PDGF-BB antibodies (1:100, orb101747, Biorbyt, Cam-
bridge, UK). Endogenous peroxidase was blocked with 0.3% (v/v)
H2O2 for 10 min. After washing in PBS containing 0.1% Tween 20,
the slides were reacted with horseradish peroxidase-conjugated
goat anti-mouse/rabbit secondary antibody (GK500711, Genetech,
Shanghai, China) for 1 h at room temperature according to the
manufacturer’s instructions.
2.10. Fertility test
A total of 60 rats (120 uterine horns) were used in the fertility
test. The right uterine horn of each rat was treated as described
above and assigned randomly to the CS/UC-MSCs group (n = 20
uterine horns), the CS group (n = 20 uterine horns) or the NR group
(n = 20 uterine horns). The left uterine horn of each rat was left
intact as the control group (Con) (n = 60 uterine horns). At day
60 post-treatment, the rats were mated with male SD rats for
4 days. The first day of mating was regarded as gestational day 0.
At day 18–20 after gestation, the uteri were exposed under anes-
thesia to confirm the presence of embryos, and the embryos larger
than 1 cm in diameter were counted.
2.11. Statistical analysis
Two independent observers performed endometrial thickness
measurement, and the average was used for subsequent analysis.
163
The percentage positive area in histological and immunohistologi-
cal staining was measured using Image-J software (NIH, USA). Data
were presented as means ± standard deviation. Multiple compar-
isons were conducted using one-way analysis of variance (SPSS
17.0). The
2 test was used for analyzing the results of fertility
tests. Statistical significance was assumed for p < 0.05 (* and **
indicate p < 0.05 and p < 0.01, respectively).
3. Results
3.1. Characterization of CS/UC-MSCs
After thawing, UC-MSCs exhibited a shuttle-like morphology.
UC-MSCs were positive for CD73, CD90, and CD105, and negative
for CD34, CD45, CD19, CD11b, and HLA-DR. UC-MSCs were able
to differentiate into osteoblasts, adipocytes and chondrocytes
(Fig. S1A, B, D).
The macroscopic appearance and microscopic structure of CSs
with or without seeding with UC-MSCs were displayed in Fig. 1.
Before seeding with UC-MSCs, the CS showed a sheet-like mor-
phology (Fig. 1A). HE staining and SEM showed that the CS had a
porous structure with a pore size of 100–200 mm (Fig. 1B, C). There
were no obvious differences between the gross views of the CS and
the CS/UC-MSCs after culture in complete medium (red color) for
12 h (Fig. 1D). As shown by HE staining, UC-MSCs attached well
to the CS (Fig. 1E). A large number of cells with shuttle-like mor-
phology were observed on the CS/UC-MSCs surface by SEM
(Fig. 1, F). The CCK-8 assay demonstrated that the UC-MSCs prolif-
erated well on both CS and TCPS. Moreover, after 72 h incubation,
cells on the CS showed higher proliferative activity than those on
TCPS, which may be attributed to the three-dimensional structure
of the CS (Fig. 1, G) (n = 3).
3.2. Paracrine effects of CS/UC-MSCs on HESCs
The paracrine effects of CS/UC-MSCs on the proliferation and
apoptosis of HESCs were evaluated using a trans-well model. HESCs
were positive for vimentin and negative for cytokeratin, and the
purity of HESCs was nearly 100% (Fig. S1, C). As shown in Fig. 1H,
the results of the CCK-8 assay demonstrated that the viabilities
of HESCs cultured in different conditions increased with culture
time, indicating that all these cells could proliferate under the
three conditions tested. However, three different proliferation pro-
files were observed. At days 1 and 2, the three groups showed sim-
ilar CCK-8 values. After culturing for 3 days, the CCK-8 values of the
HESCs in the CS/UC-MSCs group (0.90 ± 0.04) were higher than
those of the negative control group (0.60 ± 0.09) (p < 0.05, n = 3),
albeit still lower than those of the positive control group
(1.10 ± 0.04) (p < 0.05, n = 3). Analysis of HESC apoptosis under dif-
ferent culture conditions is shown in Fig. 1, I. Co-culture with CS/
UC-MSCs decreased the rate of HESC apoptosis (31 ± 4%) after
48 h compared with the negative control group (62 ± 2%)
(p < 0.01, n = 3) (Fig. 1, I). No significant difference was found
between the CS/UC-MSCs group and the positive control group.
Higher levels of VEGF-A, TGF-b1, and PDGF-BB in the super-
natants of co-cultures from the CS/UC-MSCs group were detected
by ELISA than in those from the negative control group (Fig. 1J, K,
L). The concentration of VEGF-A in culture supernatants from the
CS/UC-MSCs group (440 ± 50 pg/mL) was higher than that in the
negative control group (15.2 ± 0.9 pg/mL) (p < 0.01, n = 4). The con-
centration of TGF-b1 in culture supernatants from the CS/UC-MSCs
group (84 ± 4 pg/mL) was higher than that in the negative control
group (66 ± 5 pg/mL) (p < 0.05, n = 4). The concentration of PDGF-
BB in culture supernatants from the CS/UC-MSCs group
164 L. Xin et al. / Acta Biomaterialia 92 (2019) 160–171

Fig. 1. Characterization and paracrine effects of CS/UC-MSCs. (A, D) Macroscopic observation of CS (A) and CS/UC-MSCs (D). (B, E) HE staining of CS (B) and CS/UC-MSCs (E).
(C, F) SEM images of CS (C) and CS/UC-MSCs (F). (G) CCK-8 assay of UC-MSCs cultured on CSs (CS/UC-MSCs) and TCPS (UC-MSCs) for various time periods. (H) CCK-8 assay of
HESC proliferation in a CS/UC-MSCs co-culture system. HESCs cultured in DMEM/F12 with a blank insert and HESCs cultured in DMEM/F12 containing 10% FBS were used as
negative and positive controls, respectively. (I) Statistical analysis of the percentage of Annexin V and 7-AAD positive HESCs in the CS/UC-MSCs co-culture system measured
by flow cytometric analysis. (J, K, L) Statistical analysis of the concentrations of VEGF-A(J), TGF-b1(K), and PDGF-BB(L) in the CS/UC-MSCs co-culture system measured by
ELISA.

(36 ± 8 pg/mL) was higher than that in the negative control group (Fig. 2 B1 and B5). With longer time post-transplantation, the num-
(5.4 ± 0.4 pg/mL) (p < 0.05, n = 4). ber of CM-Dil-labeled UC-MSCs decreased (Fig. 2 B2–B4).

3.3. In vivo tracing of CS/UC-MSCs 3.4. Gross views of harvested uteri

As shown in Fig. S2, a rat model of endometrial damage was
established via a scraping treatment and demonstrated by histo-
logical staining. The stromal layer of the damaged endometrium
became thinner compared to a normal endometrium with a com-
plete luminal epithelial layer and stromal layer. Moreover,
immunohistochemical analysis showed that pan-cytokeratin posi-
tive epithelial cells were found on the normal endometrial luminal
surface, but no positive cells were observed following scraping
treatment (Fig. S2C–F).
To track the transplanted CS/UC-MSCs in vivo, HE staining of
uteri was performed at each time interval (Fig. 2, A). At 1 and
3 days post-transplantation, CSs with porous structures could still
be observed in the wound region (Fig. 2 A1, A2). At 5 and 7 days
post-transplantation, no obvious porous structure was observed,
probably due to the fusion of the CS with surrounding tissues
(Fig. 2 A3, A4).
To track the transplanted UC-MSCs in the regenerated endome-
trium, immunofluorescence of uteri at each time interval was ana-
lyzed (Fig. 2, B). At 3 days post-transplantation, a number of CM-
Dil-labeled UC-MSCs were observed in the wounded endometrium
At day 15 post-surgery, hydrometra and structural deformation
of the wounded sections were observed in the NR group (Fig. 3, B)
and the CS group (Fig. 3, C). However, the CS/UC-MSCs group
exhibited hydrometra only at the ends of uteri, as well as mild
hyperemia and edema (Fig. 3, D). At 30 days post-transplantation,
severe stenosis of the uterine cavity near the wounded section
was observed in the NR group (Fig. 3, F) and the CS group (Fig. 3,
G). By contrast, in the CS/UC-MSCs group, hyperemia was observed
at the lesion site without uterine atrophy (Fig. 3, H). At 60 days
post-transplantation, normal-seeming uteri were harvested in the
CS/UC-MSCs group (Fig. 3, L), similar to those of the sham group
(Fig. 3 A, E, I). However, the uteri of the CS group and the NR group
showed severe uterine atrophy (Fig. 3, J, K).
3.5. HE and Masson’s trichrome staining
Restoration of the endometrium following different treatments
was assessed by HE staining. At 15 days post-surgery, the NR group
and the CS group exhibited endometrial regeneration without
luminal structures (Fig. 4 A2, A3). However, histological observa-
 L. Xin et al. / Acta Biomaterialia 92 (2019) 160–171 165

Fig. 2. Tracing of the implanted CS/UC-MSCs in vivo. (A) HE staining of uteri after CS/UC-MSCs transplantation for 1 day (A1), 3 days (A2), 5 days(A3) and 7 days (A4). (B)
Immunofluorescence of uteri after CS/UC-MSCs transplantation for 3 days (B1, B5), 7 days (B2), 15 days (B3) and 30 days (B4). UC-MSCs were labeled with CM-Dil.

Fig. 3. Morphology of uteri following different treatments for 15 days (A–D), 30 days (E–H) and 60 days (I–L). Treatments included the sham group (Sham) (A, E, I), the natural
repair group (NR) (B, F, J), the CS group (CS) (C, G, K) and the CS/UC-MSCs group (CS/UC-MSCs) (D, H, L).

tion of the CS/UC-MSCs group showed endometrial regeneration
with apparent luminal structures (Fig. 4, A4). The thickness of
the regenerated endometrium in the CS/UC-MSCs group
(710 ± 60 mm) was significantly greater than that in the NR group
(560 ± 20 mm) (p < 0.01, n = 6) and the CS group (530 ± 10 mm)
(p < 0.01, n = 6) (Fig. 4 A2, A3). At 30 days post-surgery, the struc-
ture of the endometrium appeared well organized with luminal
epithelium and secretory glands in the stromal layer of the CS/
UC-MSCs group (Fig. 4, A8) By contrast, no luminal structures
and disordered glands were observed in the NR group and the CS
group (Fig. 4 A6, A7). The regenerated endometrium in the CS/
UC-MSCs group (620 ± 20 mm) was thicker than in the NR group
(420 ± 50 mm) (p < 0.01, n = 6) or the CS group (430 ± 30 mm)
(p < 0.01, n = 6) (Fig. 4, B). At 60 days post-transplantation, the
166 L. Xin et al. / Acta Biomaterialia 92 (2019) 160–171

Fig. 4. Effects of different treatments on endometrial regeneration and collagen remodeling. (A) HE staining of uteri after different treatments for 15 days (A1–A4), 30 days
(A5–A8) and 60 days (A9–A12) in the sham group (sham) (A1, A5, A9), the natural repair group (NR) (A2, A6, A10), the CS group (CS) (A3, A7, A11) and the CS/UC-MSCs group
(CS/UC-MSCs) (A4, A8, A12). Inserts are the corresponding overview pictures with lower magnification, and the magnified regions are marked with black squares. (B)
Statistical analysis of endometrial thickness after different treatments for 15 days, 30 days and 60 days. (C) Collagen staining of uteri using Masson trichrome after different
treatments for 15 days (C1–C4), 30 days (C5–C8) and 60 days (C9–C12) in the sham group (sham) (C1, C5, C9), the natural repair group (NR) (C2, C6, C10), the CS group (CS)
(C3, C7, C11) and the CS/UC-MSCs group (CS/UC-MSCs) (C4, C8, C12). Inserts are the corresponding overview pictures with lower magnification, and the magnified regions are
marked with black squares. (D) Statistical analysis of the percentages of collagen positive staining after different treatments for 15 days, 30 days and 60 days.

CS/UC-MSCs group showed normal-seeming endometrium (Fig. 4,
A12). However, the NR group and the CS group exhibited severe
IUAs (Fig. 4 A10, A11). The thickness of the endometrium in the
CS/UC-MSCs group (610 ± 30 mm) was greater than that in the NR
group (170 ± 20 mm) (p < 0.01, n = 6) and the CS group
(220 ± 20 mm) (p < 0.01, n = 6) (Fig. 4, B).
To evaluate collagen remodeling in the reconstructed endome-
trium after transplanting CS/UC-MSCs, Masson’s trichrome stain-
ing was performed (Fig. 4, C) and areas of collagen staining were
analyzed quantitatively. At 15 days post-transplantation, the CS/
UC-MSCs group (43 ± 3%) showed lower collagen deposition than
the NR group (70 ± 3%) (p < 0.01, n = 6) and the CS group
(62 ± 3%) (p < 0.01, n = 6) (Fig. 4 C2–C4, D). At 30 days post-
transplantation, collagen deposition was severe in the NR group
and the CS group. Lower collagen staining was detected in the
CS/UC-MSCs group (38 ± 5%) compared with the NR group
(73 ± 2%) (p < 0.01, n = 6) and the CS group (74 ± 2%) (p < 0.01,
n = 6) (Fig. 4 C6–C8, D), reflecting mild fibrosis of wounds treated
with CS/UC-MSCs. At 60 days post-transplantation, dense endome-
trial fibrosis was confirmed in the NR group and CS group (Fig. 4
C10, C11). However, collagen deposition in the CS/UC-MSCs group
(41 ± 2%) was similar to that of the sham group (41 ± 3%) (p greater
than 0.05, n = 6), and much lower than that of the NR group
(75 ± 2%) (p < 0.01, n = 6) and the CS group (74 ± 2%) (p < 0.01,
n = 6) (Fig. 4 C9–C12, D).
3.6. Immunohistochemical staining of Ki67 and pan-cytokeratin
Studies have shown that the initial repair of endometrial
epithelium is associated with shedding of endometrial tissue dur-
ing the menstrual phase [42]. Early rapid re-epithelialization is
critical for subsequent endometrial recovery after endometrial
damage. Ki67 and cyto-keratin are markers of cellular proliferation
and epithelium recovery, respectively (Fig. 5).
The number of Ki67-positive cells increased in all groups as
time elapsed after surgery. However, at 15 days post-
transplantation, the CS/UC-MSCs group showed significant upregu-
lation in the percentage of Ki67 positive areas (15 ± 2%) (Fig. 5 A4,
B) compared with the NR group (5.9 ± 0.8%) (p < 0.01, n = 6) (Fig. 5
A2, B) and the CS group (6.7 ± 0.6%) (p < 0.01, n = 6) (Fig. 5 A3, B).
These proliferating cells were mainly located in the shallow layer
of the endometrium (Fig. 5 A4, B). At day 30, a higher percentage
of Ki67 positive areas were observed in the CS/UC-MSCs group
(17 ± 2%) (Fig. 5 A8, B) compared with the NR group (9 ± 2%)
(p < 0.01, n = 6) (Fig. 5 A6, B) and the CS group (11 ± 2%)
(p < 0.01, n = 6) (Fig. 5 A7, B). At day 60, the percentage of Ki67 pos-
itive areas in the CS/UC-MSCs group (21 ± 2%) (Fig. 5 A12, B) was
similar to that in the Sham group (24 ± 2%) (p > 0.05, n = 6)
(Fig. 5 A1, A5, A9, B) but was still higher than that in the NR group
(9 ± 1%) (p < 0.01, n = 6) (Fig. 5 A10, B) and the CS group (11 ± 1%)
(p < 0.01, n = 6) (Fig. 5 A11, B).
The number of pan-cytokeratin positive cells increased in the
CS/UC-MSCs group over time after surgery. However, the number
of pan-cytokeratin positive cells in the NR and CS groups remained
low (Fig. 5 C, D). At 15 days post-transplantation, the CS/UC-MSCs
group (20 ± 1%) (Fig. 5 C4, D) showed obvious upregulation in the
percentage of pan-cytokeratin positive areas compared with the
NR group (4 ± 1%) (p < 0.01, n = 6) (Fig. 5 C2, D) and the CS group
(2 ± 1%) (p < 0.01, n = 6) (Fig. 5 C3, D). At 30 days post-
transplantation, the percentage of pan-cytokeratin positive areas
 L. Xin et al. / Acta Biomaterialia 92 (2019) 160–171 167

Fig. 5. Effects of different treatments on cell proliferation and epithelial recovery. (A) Immunohistochemical staining of Ki67 for cell proliferation after different treatments
for 15 days (A1–A4), 30 days (A5–A8) and 60 days (A9–A12) in the sham group (sham) (A1, A5, A9), the natural repair group (NR) (A2, A6, A10), the CS group (CS) (A3, A7, A11)
and the CS/UC-MSCs group (CS/UC-MSCs) (A4, A8, A12). Inserts are the corresponding overview pictures with lower magnification, and the magnified regions are marked with
black squares. (B) Statistical analysis of the percentages of Ki67 positive areas after different treatments for 15 days, 30 days and 60 days. (C) Immunohistochemical staining
of pan-cytokeratin after different treatments for 15 days (C1–C4), 30 days (C5–C8) and 60 days (C9–C12) in the sham group (sham) (C1, C5, C9), the natural repair group (NR)
(C2, C6, C10), the CS group (CS) (C3, C7, C11) and the CS/UC-MSCs group (CS/UC-MSCs) (C4, C8, C12). Inserts are the corresponding overview pictures with lower
magnification, and the magnified regions are marked with black squares. (D) Statistical analysis of the percentages of pan-cytokeratin positive areas after different treatments
for 15 days, 30 days and 60 days.

in the CS/UC-MSCs group (24 ± 2%) (p < 0.01, n = 6) (Fig. 5 C8, D)
was higher than that in the NR group (4 ± 1%) (p < 0.01, n = 6)
(Fig. 5 C6, D) and the CS group (4 ± 2%) (p < 0.01, n = 6) (Fig. 5 C7,
D). At day 60, the CS/UC-MSCs group (26 ± 1%) (Fig. 5 C12, D)
had similar numbers of pan-cytokeratin positive areas as the SHAM
group (33 ± 2%) (p > 0.05, n = 6) (Fig. 5 C1, C5, C9, D), and both were
significantly higher than the NR group (5 ± 2%) (p < 0.01, n = 6)
(Fig. 5 C10, D) and the CS group (4 ± 1%) (p < 0.01, n = 6) (Fig. 5
C11, D).
3.7. Immunohistochemical staining of ERa and PR
Following endometrial re-epithelialization, the functional layer
of endometrium increased in size and differentiated under the
influence of circulating estrogen and progesterone. The expression
of ERa and PRs is critical for endometrial regeneration.
As shown in Fig. 6, ERa positive cells were mainly located in the
epithelium (Fig. 6, A). At 15 days post-transplantation, the CS/UC-
MSCs group showed obvious upregulation in the percentage of
ERa positive areas (13 ± 2%) (Fig. 6 A4, B) compared with that in
the NR group (2.1 ± 0.4%) (p < 0.01, n = 6) (Fig. 6 A2, B) and the
CS group (2.7 ± 0.7%) (p < 0.01, n = 6) (Fig. 6 A3, B). At 30 days
post-transplantation, the percentage of ERa positive areas in the
CS/UC-MSCs group (13 ± 1%) (p < 0.01, n = 6) (Fig. 6 A8, B) was
higher than that in the NR group (2.4 ± 0.6%) (p < 0.01, n = 6)
(Fig. 6 A6, B) and the CS group (3.4 ± 0.6%) (p < 0.01, n = 6) (Fig. 6
A7, B). At day 60, the CS/UC-MSCs group (15.3 ± 0.9%) (Fig. 6 A12,
B) showed a significant increase in the percentage of ERa positive
areas compared with the NR group (5 ± 1%) (p < 0.01, n = 6) (Fig. 6
A10, B) and the CS group (4 ± 1%) (p < 0.01, n = 6) (Fig. 6 A11, B).
The results of PR expression analysis were similar to those for
ERa. PR positive cells were mainly located in the epithelium
(Fig. 6, C). At 15 days post-transplantation, the CS/UC-MSCs group
showed upregulation in the percentage of PR positive areas
(12 ± 1%) (Fig. 6 C4, D) compared with the NR group (3.4 ± 0.6%)
(p < 0.01, n = 6) (Fig. 6 C2, D) and the CS group (3.1 ± 0.5%)
(p < 0.01, n = 6) (Fig. 6 C3, D). At 30 days post-transplantation,
the percentage of PR positive areas in the CS/UC-MSCs group
(17 ± 1%) (p < 0.01, n = 6) (Fig. 6 C8, D) was higher than in the NR
group (4.7 ± 0.7%) (p < 0.01, n = 6) (Fig. 6 C6, D) and the CS group
(4.2 ± 0.6%) (p < 0.01, n = 6) (Fig. 6 C7, D). At day 60, the numbers
of PR-positive areas in the CS/UC-MSCs group (19 ± 2%) (Fig. 6
C12, D) were similar to those of the Sham group (23 ± 3%)
(p > 0.05, n = 6) (Fig. 6 C1, C5, C9, D), and both were significantly
higher than the NR group (4.9 ± 0.7%) (p < 0.01, n = 6) (Fig. 6 C10,
D) and the CS group (4.5 ± 0.5%) (p < 0.01, n = 6) (Fig. 6 C11, D).
3.8. Immunohistochemical staining of VEGF-A, TGF-b1, and PDGF-BB
Growth factors participate in endometrial regeneration via
autocrine and/or paracrine effects between endometrial stromal
cells and epithelial cells. They are also involved in mediating the
effects of estrogen on proliferation and progesterone on differenti-
ation [43]. Immunohistochemical staining for VEGF-A, TGF-b1, and
PDGF-BB was performed at 15 days post-surgery (Fig. S3).
168 L. Xin et al. / Acta Biomaterialia 92 (2019) 160–171

Fig. 6. Effects of different treatments on expression of ERa and PR. (A) Immunohistochemical staining of ERa after different treatments for 15 days (A1–A4), 30 days (A5–A8)
and 60 days (A9–A12) in the sham group (sham) (A1, A5, A9), the natural repair group (NR) (A2, A6, A10), the CS group (CS) (A3, A7, A11) and the CS/UC-MSCs group (CS/UC-
MSCs) (A4, A8, A12). Inserts are the corresponding overview pictures with lower magnification, and the magnified regions are marked with black squares. (B) Statistical
analysis of the percentages of ERa positive areas after different treatments for 15 days, 30 days and 60 days. (C) Immunohistochemical staining of PR after different
treatments for 15 days (C1–C4), 30 days (C5–C8) and 60 days (C9–C12) in the sham group (sham) (C1, C5, C9), the natural repair group (NR) (C2, C6, C10), the CS group (CS)
(C3, C7, C11) and the CS/UC-MSCs group (CS/UC-MSCs) (C4, C8, C12). Inserts are the corresponding overview pictures with lower magnification, and the magnified regions are
marked with black squares (D) Statistical analysis of the percentages of PR positive areas after different treatments for 15 days, 30 days and 60 days.

VEGF-A, TGF-b1, and PDGF-BB positive cells were mainly
located in the epithelium. The CS/UC-MSCs group showed obvious
upregulation in the percentage of VEGF-A positive areas (20 ± 4%)
(Fig. S3 A4, B) compared with the NR group (10 ± 1%) (p < 0.05,
n = 6) (Fig. S3 A2, B) and the CS group (8 ± 2%) (p < 0.05, n = 6)
(Fig. S3 A3, B). Similarly to VEGF-A, TGF-b1 expression in the CS/
UC-MSCs group (10 ± 1%) (Fig. S3 A8, C) was higher than in the
NR group (4 ± 2%) (p < 0.05, n = 6) (Fig. S3 A6, C) and the CS group
(3 ± 1%) (p < 0.05, n = 6) (Fig. S3 A7, C). Expression of PDGF-BB in
the CS/UC-MSCs group (14 ± 3%) (Fig. S3 A12, D) was also higher
than in the NR group (5 ± 1%) (p < 0.05, n = 6) (Fig. S3 A10, D)
and the CS group (5 ± 1%) (p < 0.05, n = 6) (Fig. S3 A11, D).
3.9. Fertility restoration
At gestational day 18–20, pregnant uteri and the corresponding
embryos were collected and the results are displayed in Fig. 7. The
pregnancy rate and the rate of implantation of embryos with diam-
eters larger than 1 cm were summarized in Table 1. In the control
group, the pregnancy rate and the embryo implantation rate was
95.0% and 90.4%, respectively. In the NR group, no implanted
embryos were observed (Fig. 7, C). In the CS group, only one preg-
nancy was observed among 20 uteri, and only one embryo larger
than 1 cm was identified (Fig. 7 D, E). However, in the CS/UC-
MSCs group, the pregnancy rate was 45.0% and the rate of embryo
implantation was 30.3% (Fig. 7 F, G and Table 1). The CS/UC-MSCs
group exhibited improved fertility compared with the NR group
and the CS group. These results implied that the CS/UC-MSCs could
regenerate functional endometrium for the implantation of
embryos and promote fertility restoration.
4. Discussion
In this study, we fabricated a CS/UC-MSCs and demonstrated
that the scaffold facilitated endometrial regeneration and fertility
restoration. Thicker endometrium, obvious luminal structures,
and better collagen remodeling were observed in the CS/UC-
MSCs group. These findings could be attributed to the proliferation
of intrinsic endometrial cells and epithelial reconstruction.
Normally, IUAs are characterized by severe fibrosis and lack of
normal endometrium. Hysteroscopy and subsequent intrauterine
physical barriers are the main conventional methods used clini-
cally to treat IUAs. These intrauterine devices may prevent re-
adhesion to some degree. However, due to low biocompatibility
and amenorrhea associated with compression of the endometrium,
they may cause inflammatory reactions and be harmful for
endometrial regeneration [44]. Therefore, a degradable and active
scaffold is expected to overcome these limitations.
In our study, we used a CS loaded with stem cells, which has
proven efficacy in tissue regeneration [29–31]. We fabricated the
CS by freeze-drying and thermal treatment crosslinking. The scaf-
fold exhibited a porous structure, which was suitable for cell adhe-
sion and nutrient or oxygen delivery. As UC-MSCs exhibit many
advantages including their non-invasive collection, high prolifera-
tion potential, and mutilineage differentiation, these cells have
 L. Xin et al. / Acta Biomaterialia 92 (2019) 160–171
Fig. 7. Effects of different treatments on fertility restoration. Gross views of pregnant uteri and corresponding embryos from the control group (Con) (A, B), the CS group (CS)
(D, E) and the CS UC-MSCs group (CS/UC-MSCs) (F, G). (C) Gross view of uteri from the natural repair group (NR).
Table 1
Reproductive outcomes over 60 days following different treatments.
Variable
Total number of uterine horns
Number (percentage) of pregnant uterine horns
Total number of embryos
Number (percentage) of embryos larger than 1 cm
a
p < 0.01 NR group versus CS/UC-MSCs group.
b
p < 0.01 CS group versus CS/UC-MSCs group.
c
p < 0.01 NR group versus CS/UC-MSCs group.
d
p > 0.05 CS group versus CS/UC-MSCs group.
been regarded as an ideal source for the fabrication of CS/stem cell
constructs [45–49]. We showed that the porous CS provided an
ideal physical support for the attachment and proliferation of
UC-MSCs.
In vitro, we established a co-culture system and showed that the
CS/UC-MSCs promoted HESC proliferation and inhibited apoptosis.
Similarly, many studies have also demonstrated that UC-MSCs
inhibited apoptosis, promoted angiogenesis, and modulated
immune responses by secreting a variety of soluble factors, includ-
ing VEGF-A, insulin-like growth factor 1, TGF-b, and hepatocyte
growth factor [50–53]. In the present study, higher levels of
VEGF-A, TGF-b1, and PDGF-BB were observed in the CS/UC-MSCs
group compared with the negative control group. From these
results, we concluded that the proliferation and decreased apopto-
sis of HESCs might relate to paracrine effects of CS/UC-MSCs. It
should be pointed out that immune modulation of MSCs might
be another important effect involved in endometrial regeneration
and should be investigated further. We found in vivo that the site
adjacent to CS/UC-MSCs transplantation in uteri displayed higher
levels of VEGF-A, TGF-b1, and PDGF-BB compared with the NR
group and the CS group. These growth factors have important roles
in endometrial regeneration [54]. VEGF is vital for endometrial re-
169
Control NR CS CS/UC-MSCs
60 20 20 20
57 (95.0%) 0 (0)a 1 (5.0%)b 9 (45.0%)
417 0 3 33
377 (90.4%) 0 (0)c 1 (33.3%)d 10 (30.3%)
epithelialization and vascularization [55]. TGFbs are suggested to
induce proliferation and regulate immune responses in the endo-
metrium [56,57]. PDGFs promote stromal cell proliferation via
autocrine effects at the proliferative stage [54,58].
In animal experiments, we developed an endometrium scraping
model in rats and confirmed that scraping ultimately led to severe
IUAs and infertility. This model was quite similar to the induction
of IUAs in humans, and this model may be useful in further
research. By tracing the CS/UC-MSCs in vivo, no obvious porous
material was observed at 7 days post-transplantation. Similarly,
we observed that the intensity of labeled UC-MSCs in uteri was
high during the first 7 days post-transplantation and then faded
over time. Thus, CS/UC-MSCs transplantation might create an
appropriate environment for tissue repair during the early stages
following endometrial damage.
We observed normal morphology, thicker endometrium and
better collagen remodeling after CS/UC-MSCs transplantation. By
contrast, atrophied uteri, thin endometrium and severe collagen
deposition were observed in the NR and CS groups.
Re-epithelialization of endometrium is critical for scar-free
repair with menstrual breakdown in humans. Epithelial repair
may be associated with resurfacing of epithelial cells from glands
170 L. Xin et al. / Acta Biomaterialia 92 (2019) 160–171
in the basal layer [59]. In our study, we demonstrated that the CS/
UC-MSCs increased cell proliferation in the endometrium by Ki67
immunostaining, similar to the research of bone mesenchymal
stem cells(BMSCs)’ promoting proliferation effect observed in mur-
ine uterus after IUA [26]. These proliferating cells on the luminal
surface were positive for pan-cytokeratin, which indicated better
epithelium recovery following CS/UC-MSCs transplantation.
After re-epithelialization, ERa and PR expression is induced in
endometrial cells by rising estrogen and progesterone levels. As a
result, regeneration of endometrial mucosa and revascularization
occurs in the proliferative stage [60–62]. ERa and PR, as markers
of endometrial receptivity, are critical targets for clinical IUA treat-
ment. Due to the lack of functional endometrium in IUAs, the
effects of estrogen and progesterone supplement therapy are not
satisfactory. In this study, the CS/UC-MSCs remarkably upregulated
the expression of ERa and PR. Moreover, ERa and PR were mainly
expressed in the regenerated epithelium. These results indicated
that the regeneration of endometrial glands and luminal epithe-
lium played a vital role in recovery of endometrial function. Estro-
gen or progesterone could bind to ERa or PR, which activated
downstream signal transduction and triggered the expression to
maintain uterine function [63]. These results implicated that sup-
plementation with estrogen and progesterone following CS/UC-
MSCs transplantation might lead to better outcomes in clinical
treatment.
We showed that endometrial thickness and collagen deposition
did not change significantly from day 15 to day 60 post-
transplantation, indicating that primary structural remodeling
was almost complete within 15 days. However, the gradually
increased expression of Ki67, pan-cytokeratin, ERa, and PR as time
elapsed post-transplantation probably indicated that the recovery
of precise structures (such as the glandular epithelium of the endo-
metrium) and their functions may require longer time. Therefore,
we performed the fertility test at 60 days post-surgery. The preg-
nancy capacity is the gold standard to confirm the function of
regenerated endometrium. We found that pregnancy rates in the
CS/UC-MSCs group were higher than in the CS group and the NR
group, indicating the functional recovery of regenerated endome-
trium after transplantation with CS/UC-MSCs. Early implantation
sites can be evaluated using implantation-specific assay such as
Evans-blue staining on gestational day 4.5–5.5, but embryonic
development is still unclear during pregnancy. In this study,
embryos were observed on gestational day 18–20 and those larger
than 1 cm were deemed as well-developed embryos. Our results
indicated that the rate of implantation of embryos larger than
1 cm in the CS/UC-MSCs group was higher than in the NR group,
which can be attributed to the better-organized epithelium and
improved responsiveness to sex hormones of the regenerated
endometrium in the CS/UC-MSCs group.
5. Conclusions
A CS/UC-MSCs was fabricated and applied for endometrial
regeneration. In vitro studies showed that the CS/UC-MSCs pro-
moted HESC proliferation and inhibited apoptosis through para-
crine effects. The transplanted CS/UC-MSCs maintained luminal
structures, and promoted endometrium regeneration and collagen
remodeling in a rat model of endometrial damage. The regenerated
endometrium expressed ERa and PR and the ability of the regener-
ated endometrium to receive embryos was confirmed. Together,
these results indicated that the CS/UC-MSCs could facilitate the
reconstruction of endometrium structure and function. The topical
transplantation of CS/UC-MSCs after trans-cervical resection of
adhesions offers a novel strategy to prevent re-adhesion, promote
endometrium regeneration and improve pregnancy outcomes in
the clinic.
Acknowledgments
The authors thank Boyalife Group Ltd. for help with flow cytometry
and multipotent differentiation analysis of UC-MSCs. This study
was financially supported by the National Key Research Program
of China (2018YFC1004800, 2017YFA0104900), Wu Jieping medi-
cal fund (320.6755.15028), the National Natural Science Founda-
tion of China (51673167), and the Key Research and
Development Program of Zhejiang Province (2017C03022).
Author disclosure statement
The authors have no conflicts of interest to declare.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.actbio.2019.05.012.
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