A collagen scaffold loaded with human umbilical cord-derived mesenchymal stem cells facilitates endometrial regeneration and restores fertility

Acta Biomaterialia 92 (2019) 160–171

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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 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 b

a r t i c l e

i n f o

Article history: Received 22 November 2018 Received in revised form 17 April 2019 Accepted 6 May 2019 Available online 7 May 2019 Keywords: Collagen scaffold Umbilical cord-derived mesenchymal stem cells Endometrial regeneration Intrauterine adhesion Fertility restoration

a b s t r a c t In women of reproductive age, severe injuries to the endometrium are often accompanied by endometrial scar formation or intrauterine adhesions (IUAs), which can result in infertility or miscarriage. Although many approaches have been used to treat severe IUAs, high recurrence rates and endometrial thinning have limited therapeutic efficiency. In this study, a collagen scaffold (CS) loaded with human umbilical cord-derived mesenchymal stem cells (UC-MSCs) was fabricated and applied for endometrial regeneration. The CS/UC-MSCs promoted human endometrial stromal cell proliferation and inhibited apoptosis in vitro through paracrine effects. In a model of endometrial damage, transplantation with the CS/UCMSCs maintained normal luminal structure, promoted endometrial regeneration and collagen remodeling, induced intrinsic endometrial cell proliferation and epithelium recovery, and enhanced the expression of estrogen receptor a and progesterone receptor. An improved ability of the regenerated endometrium to receive embryos was confirmed. Together, our results indicate that the CS/UC-MSCs promoted 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 regeneration. Herein, UC-MSCs, known for low immunogenicity and high proliferative potential, exhibit promising 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 strategy for patients suffering severe intrauterine adhesion and infertility. Ó 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

⇑ Corresponding authors at: Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, No. 3 Qingchun East Road, Jianggan District, Hangzhou 310016, China. E-mail addresses: [email protected] (L. Ma), [email protected] (S. Zhang). 1 These authors contributed equally. https://doi.org/10.1016/j.actbio.2019.05.012 1742-7061/Ó 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Human endometrium is a dynamic and regenerative tissue composed of epithelial cells, stromal cells, vascular smooth cells, and vascular endothelial cells. The endometrium can be structurally divided into two zones: the upper functional layer and lower basal layer. During a menstrual cycle, the functional layer is shed while the permanent basal layer regenerates a brand-new

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functional layer according to fluctuating levels of estrogen and progesterone. However, intrauterine adhesions (IUAs), which result from repeated curettage, infections and intrauterine operations, can cause damage to the endometrial basal layer [1,2]. Subsequent failure of re-epithelization, inflammation and fibrotic tissue deposition lead to shrinking of the uterine cavity and endometrium, inactive glands and poorly vascularized stroma. A meta-analysis showed that approximately 19% of women experiencing miscarriages were diagnosed IUAs within 12 months. Among these women, 58.1%, 28.2% and 13.7% had mild, moderate and severe IUAs, respectively [1]. Importantly, severe IUAs may be associated with infertility, including menstrual disorders, spontaneous abortion and pregnancy complications [3,4]. Many strategies, including hysteroscopy, hormone therapy, intrauterine devices and antibiotics, have been used for treatment of IUAs [5–7]. However, endometrial regeneration remains a significant challenge. Current treatments can temporarily normalize the shape and volume of the uterine cavity but cannot restore the structure and function of the endometrium, leading to IUA reformation. The recurrence 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]. Multiple approaches have been proposed to prevent recurrence of IUAs after surgery, such as application of hyaluronic acid gel in humans [10], transplantation of freeze-dried amnion grafts in humans [11], or transplantation of oral mucosal epithelial cell sheets in rats [12]. These interventions can prevent the recurrence of IUAs to some degree, but do not restore a functional endometrium. Therefore, the question of how to reconstruct an endometrium with normal morphology and function is a vital issue for IUA treatment. Several medications such as aspirin [13–15] and granulocyte colony stimulating factor [16] are recommended to promote human endometrial regeneration by increasing blood perfusion. However, due to damaged revascularization and ischemia in severe IUA lesions, the clinical effects of these medications are limited [17]. It has become evident that endometrial stem/progenitor cells in both the functional and basal layers are responsible for endometrial regeneration [18–20]. In IUAs, the numbers and function of endometrial stem/progenitor cells are diminished due to the lack of functional endometrium [3]. Recently, cell therapy toward uterus have been popular in treating endometrial dysfunction [20]. Mesenchymal stem cells (MSCs) have emerged as a promising cell type for tissue regeneration since they are characterized by multipotent differentiation, high expansive potential, and immunomodulatory capability. Depending on the cellular source, MSCs can be classified as bone marrow-derived, umbilical cord-derived, adipose-derived, and human menstrual blood-derived MSCs. Umbilical cordderived MSCs (UC-MSCs) have been regarded as a promising source for cell-based therapies because of their easy collection, low immunogenicity, and high proliferative potential [21]. Applications of UC-MSCs include bone defects [22], renal diseases [23], and peripheral nerve injuries [24]. However, few studies have reported the application of UC-MSCs to promote endometrial repair. The main obstacle to treatment with MSCs is their low local persistence and utilization rate in the endometrium [25]. Only 0.59% or 0.65% of engrafted cells were observed around small endometrial vessels following intrauterine infusion or tail vein injection in a murine model [26]. As critical components of tissue engineering materials, porous scaffolds play an essential role in tissue repair and regeneration. Collagen, a natural biomaterial and the main component of the extracellular matrix, has been widely utilized for tissue engineering scaffolds. A collagen scaffold (CS) can provide a suitable physical support and microenvironment for transplanted stem cells [27,28]. CSs loaded with stem cells have been applied for regeneration of many tissues and organs including

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articular cartilage [29], periodontal tissue [30], and brain [31]. In the reproductive field, most studies have focused on regeneration of the uterine wall or even the entire uterus. For example, an engineered uterine tissue was constructed by encapsulating human endometrial stromal cells, epithelial cells and smooth muscle cells into a collagen/Matrigel scaffold in vitro [32]. In addition, the uterine horns of rats were reconstructed by the transplantation of stem cell-loaded CS grafts after partial excisions [33–35]. However, regeneration of an entire uterus is a major challenge because of the complexity of uterine tissue. Moreover, the primary cause of IUA is not disorder of the uterus or uterine walls, but damage to the endometrium. In this study, we aimed to develop a CS loaded with UC-MSCs for endometrial regeneration. We demonstrated that the CS/UC-MSCs promoted endometrial stromal cells proliferation and inhibited apoptosis in vitro via paracrine effects. The efficacies of the CS/UC-MSCs in restoring the structure and function of the endometrium were assessed in a rat model of endometrial damage. Finally, regenerative outcomes were assessed using a fertility test. 2. Materials and methods 2.1. UC-MSC monoculture, flow cytometric analysis and multipotent differentiation analysis Frozen UC-MSCs at passage 3 were purchased from Boyalife Group Ltd. (China). After thawing, the cells were seeded in 10-cm culture dishes (1
106 cells/dish) in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12, Gino Biological, Hangzhou, China) supplemented with 10% (v/v) fetal bovine serum (FBS, SA112.02, Cellmax, Beijing, China), 100 U/mL penicillin (Gibco), and 100 mg/mL streptomycin (Gibco). When UC-MSCs reached 80–100% confluence, cells were subcultured in quadruplicate. UC-MSCs harvested at passage 4–6 were used in this study. The phenotype and multipotent differentiation of UC-MSCs were confirmed (Fig. S1). For flow cytometric analysis, UC-MSCs (1
106) were incubated with 1 mg each of fluorescein isothiocynate(FITC)-conjugated anti-human CD73 (Biolegend, 344016, USA), phycoerythrin (PE)-conjugated anti-human HLADR (Beckman, IM1639, USA), anti-human CD34 (Beckman, A07776, USA), anti-human CD45 (Beckman, A07783, USA), antihuman CD19 (Beckman, A07769, USA), anti-human CD11b (Beckman, IM2581U, USA), PE/CF594-conjugated anti-human CD90 (Beckman, IM3703, USA), and PE/Cy7-conjugated anti-human CD105 (Biolegend, 323218, USA) antibodies for 30 min in the dark. After washing twice with phosphate-buffered saline (PBS), the samples were analyzed using a flow cytometer (Beckman Coulter, Fullerton, CA, USA). For osteogenic differentiation analysis, UCMSCs (5
103 cells/cm2) were treated with 0.1 lM dexamethasone (Sigma-Aldrich, St Louis, MO, USA), 10 mM b-glycerol phosphate (Sigma-Aldrich, St Louis, MO, USA) and 50 lg/mL ascorbic acid (Sigma-Aldrich, St Louis, MO, USA). After incubating for 15 days, positive induction was observed by Alizarin Red staining. For analysis of adipogenic and chondroblast differentiation, cells were incubated in adipogenic differentiation medium (Biological Industries, Israel) or chondrogenic differentiation medium (Biological Industries, Israel) for 15 days according to the manufacturer’s instructions. Positive induction was observed by oil red staining and Alcian Blue staining. 2.2. Human endometrial stromal cell (HESC) isolation, culture and immunofluorescence analysis HESCs were obtained from fresh endometrial specimens as previously described [36]. Informed consent was obtained from all

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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 collected 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 488conjugated goat anti-rabbit IgG (1:300, ab150077, Abcam, Cambridge, 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 , 6diamidino-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 sectioned 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 electron microscopy (SEM, Hitachi Model S-520, Japan). The SEM specimens were fixed with 2.5% (v/v) glutaraldehyde for 24 h at 4 °C, post-fixed with 1% osmium tetroxide, dehydrated in graded ethanol, 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).

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 proliferation 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/UCMSCs Animal experiments were approved by the committee for animal 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 Province (China) and housed in a controlled environment at 22 °C with a 12 h/12 h light/dark cycle. The animals were allowed to acclimatize 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 abdominal 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.

2.5. Effect of CS/UC-MSCs on HESC proliferation and apoptosis in vitro 2.8. In vivo tracing of implanted CS/UC-MSCs 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/

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

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uteri were harvested (n = 6). For HE staining, the uteri were fixed with 4% paraformaldehyde overnight and sectioned at 5-lm thickness. 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 fluorescence microscope (Leica, Germany).

The percentage positive area in histological and immunohistological staining was measured using Image-J software (NIH, USA). Data were presented as means ± standard deviation. Multiple comparisons 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).

2.9. Histological and immunohistological analysis

3. Results

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 transplanted 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 trichrome using conventional protocols. The thickness of the regenerated 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 performed 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 antibodies 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, Cambridge, 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, Cambridge, 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.

3.1. Characterization of CS/UC-MSCs

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 anesthesia 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.

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 morphology (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 morphology were observed on the CS/UC-MSCs surface by SEM (Fig. 1, F). The CCK-8 assay demonstrated that the UC-MSCs proliferated 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 profiles were observed. At days 1 and 2, the three groups showed similar 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 different 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 supernatants 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 concentration 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 PDGFBB in culture supernatants from the CS/UC-MSCs group

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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 (5.4 ± 0.4 pg/mL) (p < 0.05, n = 4).

(Fig. 2 B1 and B5). With longer time post-transplantation, the number 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 histological staining. The stromal layer of the damaged endometrium became thinner compared to a normal endometrium with a complete luminal epithelial layer and stromal layer. Moreover, immunohistochemical analysis showed that pan-cytokeratin positive 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 endometrium, immunofluorescence of uteri at each time interval was analyzed (Fig. 2, B). At 3 days post-transplantation, a number of CMDil-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-

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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 structure 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

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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 endometrium after transplanting CS/UC-MSCs, Masson’s trichrome staining 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 posttransplantation, 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 endometrial 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 posttransplantation, the CS/UC-MSCs group showed significant upregulation 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 positive 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 posttransplantation, the percentage of pan-cytokeratin positive areas

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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/UCMSCs 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 differentiation [43]. Immunohistochemical staining for VEGF-A, TGF-b1, and PDGF-BB was performed at 15 days post-surgery (Fig. S3).

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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/UCMSCs) (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 diameters 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 pregnancy was observed among 20 uteri, and only one embryo larger than 1 cm was identified (Fig. 7 D, E). However, in the CS/UCMSCs 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/UCMSCs 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 clinically to treat IUAs. These intrauterine devices may prevent readhesion 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 scaffold exhibited a porous structure, which was suitable for cell adhesion and nutrient or oxygen delivery. As UC-MSCs exhibit many advantages including their non-invasive collection, high proliferation potential, and mutilineage differentiation, these cells have

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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.

a b c d

Variable

Control

NR

CS

CS/UC-MSCs

Total number of uterine horns Number (percentage) of pregnant uterine horns Total number of embryos Number (percentage) of embryos larger than 1 cm

60 57 (95.0%) 417 377 (90.4%)

20 0 (0)a 0 0 (0)c

20 1 (5.0%)b 3 1 (33.3%)d

20 9 (45.0%) 33 10 (30.3%)

p < 0.01 p < 0.01 p < 0.01 p > 0.05

NR group versus CS/UC-MSCs group. CS group versus CS/UC-MSCs group. NR group versus CS/UC-MSCs group. 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, including 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 apoptosis 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-

epithelialization and vascularization [55]. TGFbs are suggested to induce proliferation and regulate immune responses in the endometrium [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

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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 murine 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 treatment. 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 epithelium played a vital role in recovery of endometrial function. Estrogen 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 supplementation with estrogen and progesterone following CS/UCMSCs 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 posttransplantation, 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 endometrium) and their functions may require longer time. Therefore, we performed the fertility test at 60 days post-surgery. The pregnancy 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 endometrium 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 promoted HESC proliferation and inhibited apoptosis through paracrine 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 regenerated 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.

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