Regeneration of injured endometrium using acellularized amniotic membrane loaded with adipose- derived mesenchymal stem cells in a rat model

Introduction

Intrauterine adhesion (IUA) is characterized by the formation of fibrosis. Adhesions tissues partially or completely obliterates the uterine cavity because of various damages to the basal endometrial layer [1, 2]. As a matter of fact, it usually leads to menstrual abnormalities, secondary infertilities and recurrent abortion, such serious reproductive health problems[3]. Although, with the development of hysteroscopic adhesiolysis, hormonal treatment or the placement of barriers (e.g. barrier gels, intrauterine device) these clinical methods are uses to reconstruct the anatomical structure of the uterine cavity, the high risk of re-adhesion after resection and the regeneration of functional endometrial tissue are still the major challenges[4]. Nowadays, further research on regenerative medicine has been developed to prevent adhesion recurrence of IUA[5]. Acellularized amniotic membrane (AAM) is a thin film composed of epithelial layer, basement membrane, and mesenchyme [6]. Recently, as one of the most popular natural biological materials, AAM has the outstanding advantages of low immunogenicity and high absorbability, is widely used in repairing epithelial defects such as skin, eye, abdominal wall and peritoneum, etc. [7, 8]. Other studies have also reported the value of AAM on decreasing adhesion recurrence and promoting endometrium regeneration [7].

For patients with severe IUA, when the basal layer is badly damaged, even the uterine cavity could recover by adhesiolysis, the prognosis is still poor due to the failure of functional endometrial layer regeneration[9]. Endometrial stem cells (ESCs) are considered as the source of progenitor cells that could differentiate into endometrium, and play a key role in the rapid replacement of functional layer as well [10]. According to previous reports, ESCs can differentiate into endometrial glandular epithelial cells, stromal cells and endothelial cells in vitro [10, 11]. However, ECSs are relatively hard to acquire because the sterility of cell products must meet high requirements. Therefore, it is very important to develop other replaceable, available, abundant and immune-privileged cell sources. Adipose-derived mesenchymal stem cells (ADSCs) are precursor cells that have been proved to secrete various bioactive molecules (e.g. growth factors, cytokines and chemokines) [11]. Some studies have also confirmed that ADSCs may be recruited into the uterus, where they differentiate into endometrial cells and muscle cells and angiogenesis, resulting in achieving a better pregnancy outcome [12, 13]. Besides, Shao et al confirmed this phenomenon in mouse IUA model. These reports suggest that ADSCs could migrate to the damaged endometrium and cure uterine defects [14].

However, the matter is, with the absence of supporting scaffold, stem cells usually migrate to other places after uterine implantation. Therefore, in order to avoid losing ADSCs while treating IUA, AAM should be used as a scaffold to provide support. We hypothesize that AAM loaded with ADSCs may benefit normal uterine cavity regeneration by its material biocompatibility. After 14 days’ follow-up, our results proved that AAM loaded with ADSCs could prolong the contact time with injured endometrium, reduce fibrosis formation and promote endometrium regeneration.

Result

ADSCs characteristics, AM acellularization and ADSCs/AAM compound

The ADSCs obtained from rat inguinal subcutaneous fat were cultured to the third generation. FACS analysis showed that the expression of surface markers CD29, CD44 and CD90 of ADSCs was 96.19%, 99.86% and 99.73%, respectively, whereas the expression of hematopoietic stem cell marker CD11b was 0.31%, and CD45 was 0.24% (Fig. 1A). Osteogenesis was inducted, and the calcium was brown-red with Alizarin-Red staining. Adipogenic induction of ADSCs was performed and adipocytes were identified by Oil Red O staining. Chondrogenic induction of ADSCs was performed and identified by toluidine blue staining (Fig. 1B). The ADSCs with fibroblast-like morphology were showed in Fig.2A. The fluorescence microscope showed the shape of ADSCs pre-labeled with fluorescent dye Dil (Fig. 2B). AAM presented a thin-film structure (Fig. 2C), which could be easily tailored in various shapes to fill the defect. Live/dead staining showed that ADSCs could continue to proliferate without apoptosis (Fig. 2D1 and D2). SEM showed only collagen fibers on the AAM (Fig.2E1 and E2), while the abundant ECM deposition was found in ADSCs/AAM compounds (Fig. 2F1 and F2).

The regeneration of injured endometrium

To demonstrate the specific function of ADSCs and AAM in IUA, we applied it to rat IUA models. In the sham operation group, the endometrial surface was covered with high columnar epithelial cells. In ADSCs/AAM group, at day 14 after operation, HE staining results showed intact structure of the endometrial layer as normal epithelial cells, similar with that in sham operation group. The endometrial thickness and the number of glands were higher than those in simple AAM group. However, in IUA group, the uterine cavity was completely destroyed in both the endometrium layer and myometrium layer (Fig. 3 and 4).

To evaluate the extent of endometrial fibrosis in injured endometrium in IUA rat models, we performed Masson staining of collagen fibers. In the sham group, there was almost no collagen deposition in the endometrial stroma. In the IUA group, there was a significant increase in the collagen fiber deposition compared with sham group. However, the endometrial fibrosis rate in the ADSCs/AAM group was a significant reduce compared with the IUA group at day 14 after operation (Fig. 3 and 4).

To further evaluate the regeneration of injured endometrium and the extent of fibrosis, we performed CD31, VEGF, vimentin and E-cadherin immunoexpression in all groups at day 14 after surgery (Fig.5). CD31, an endothelial cell marker presented in stroma, could be performed for measuring the microvascular density. The results showed that the microvascular density in the ADSCs/AAM group was significantly higher compared with that in the AAM group, similar with that in the sham group. VEGF, a well-known angiogenic factor presented in cytoplasm, is essential for neovascularization. The results showed that VEGF was significantly decreased in the IUA group compared with that in AAM group. After the administration of ADSCs, the level of VEGF was slightly higher, similar with the expression of VEGF in the sham group. Vimentin, a marker protein for stromal cells presented in stroma, was mainly expressed in the cytoplasm of endometrial stromal cells. The results showed that vimentin in the ADSCs/AAM group was significantly higher compared to AAM group and was similar with that in the sham group. E-cadherin is a transmembrane protein expressed in epithelial cells, and plays an important role in maintaining the stability of epithelial cells.

At last, we performed pregnancy test in 12 rats with 24 IUA models at 4 weeks after grafting with ADSCs/AAM or not (Fig.6A). In the Sham group, 4 uteri conceived (66.7%), while none of the uteri in the IUA group. In the ADSCs/AAM group, although 3 uterine conceived (50%), the average number of embryos was 1-2 embryos in each uterus, compared to 3-4 embryos in the Sham group (Fig.6B). By contrast, ADSCs/AAM led to an improved trend in fertility restoration, with a 50% pregnancy rate and 1 ± 2 gestational embryos.

The regulating function of stem cell on inflammatory response and uterine fibrosis

To further explore the regulating funtion of ADSCs during the endometrial repair, we performed the RNA sequancing of uterine tissue at 3 days and 14 days after operation. These outcomes exhibited high expression level of COL4A1, MSN and HMOX1 in IUA group compared with sham group with high expression level of C3, EEF2 and MMP7 at 3 days after surgery (Fig.7A). GO analysis showed high enrichment of endothelium development, lymphangiogenesis and leukocyte migration, which were association with the repair of injuired endothelium (Fig.7C). At 14 days, these genes including EEFLA1, IGFBP5 and RPLP0,which represented the normal endothelial funciton in sham group, exhibited high expression level compared with COL3A1, COL1A1 and ACTG2 in IUA group (Fig.7A). GO analysis exhibited high enrichment of extracellular matrix organization, cell-substrate adhesion, endoderm development and leukocyte migration, which comfirmed an endothelial adhesion (Fig.7C). Then, we compared the functional enrichment difference between AAM and ADSCs/AAM group. The outcomes exhibited high expression level of SPP1, LYZ2, APOE and CTSB in ADSCs/AAM compared with high expression level EEF1A1, COL6A1, and TAGLN in AAM group at 3 days and 14 days after surgery (Fig.7B). GO analysis confirmed the enrichment of antigen processing, leukocyte chemotaxis, phagocytosis, neutrophil activation and endocytosis in ADSCs/AAM group at 3 days after surgery. Meanwhile, at 14 days after surgery, GO analysis exhibited normal functional enrichment, including ribosome, protein targeting and RNA catabolic process in in ADSCs/AAM group (Fig.7D).

Then, we evaluated the expression of inflammatory cytokine (bFGF, IL-1β, IL-6, and TNF-α1) (Fig.8). The outcomes showed that the mRNA levels of anti-inflammatory cytokine, such as bFGF and IL-6, were significantly increased in the ADSCs/AAM group at day 3 and 7 after surgery compared to the AAM group. Meanwhile, expressions of pro-inflammatory cytokine such as TNF-α and IL-1β were significantly decreased in the ADSCs/AAM group at day 3 and 7 after surgery compared to the AAM group. However, for both anti-inflammatory cytokine and pro-inflammatory cytokine at day 14 after surgery, the expression level between ADSCs/AAM and AAM showed no significant difference.

The tracing of stem cell in vivo

ADSCs were marked with Dil, which is a flourochrome used for tracing live cells in vivo. At day 3, 7, and 14 after surgery, fluorescent microscope found that Dil-labeled ADSCs distributed close to the damaged endometrium and have incorporated into the endometrium tissue. The signal attenuated but still present at 14 days (Fig.9).

Discussion

IUA often occurred under the circumstance that endometrium fails to regenerate and fibrotic tissue developed after damage or surgery [15]. Therefore, an ideal therapeutic treatment must take both preventing fibrotic formation and promoting endometrium regeneration into consideration. In this study, we investigated the potential role of AAM loaded with ADSCs to prevent IUA after endometrial injury. Our results indicated that ADSCs/AAM could promote endometrium regeneration by measuring endometrial thickness, numbers of glands and blood vessel area. As a matter of fact, merely use AAM could help IUA recover to the normal uterine cavity, and our results suggest that the additional application of ADSCs could acheive better endometrium regeneration and reduce endometrial fibrosis.

The key of IUA treatment is to regenerate the functional endometrium, which is always achieved by the following methods: first hysteroscopic adhesiolysis to reform the cavity, then insert an intrauterine device (IUD) to prevent recurrence of fibrosis. However, due to its low biocompatibility, IUD may prevent normal endometrium regeneration but cause infection [16]. In this study, we used human AAM to regenerate endometrium. As one of the most popular biomaterials, after removing the cellular component, AAM could be used as a natural component to eliminate immunological rejections and enhance better cell adhesion, proliferation, and differentiation, making it an ideal material for regenerative medicine [17]. As we all know, intrauterine curettage at 2–4 weeks after delivery is most likely to cause scars and irreversible uterine wall damage. From our observations, AAM would gradually degrade and disappear within 7 to 14 days after implantation, which exactly happened to exert its preventive effect on the formation of IUA. Although AAM could effectively prevent fibrosis tissue caused by endometrial damage, it is still a big challenge to promote tissue regeneration, including neovascularization, cell proliferation and endometrial repair. The plus application of stem cells could help deal with the situation.

The endometrium shades and regenerates during every menstrual cycle [18]. ESCs in the basal layer are considered as the source of endometrium regeneration [19–20]. Recently, ESCs have been found in the basal layer of the human endometria, which is similar to adult mesenchymal stem cells (MSCs) [19]. As we all know, MSCs may play an important role in endometrium regeneration. Several other sources of MSCs have also been put forward for endometrium regeneration, such as bone marrow mesenchymal stromal cells (BMSCs), menstrual blood–derived mesenchymal stromal cells (mbMSCs) and amniotic tissue-derived mesenchymal stromal cells (AmMSCs) [13, 21, 22]. They all possess a strong ability in reducing tissue damage after endometrial injury. In this study, we selected ADSCs as the source. We acknowledge the main advantage of using ADSCs for its easy acquisition and quick amplification. Considering these advantages and conveniences, we finally chose ADSCs/AAM for IUA treatment in this study. One of the most important advantages of implanting ADSCs/AAM is the material could be used as a carrier to provide a feasible support for cells delivery and storage. Compared with simple AAM transplantation, ADSCs/AAM could produce thicker endometrium, more endometrial glands, and lower fibrotic area rate. Meanwhile, the research findings demonstrated that some histological changes, for instance, the high immune expression of CD31, vimentin, VEGF and E-cadherin in ADSCs/AAM group, indicating their ability to form the normal histological endometrium in IUA rat model.

It is believed that the pathogen of fibrosis is excessive ECM deposition, caused by early inflammatory activities and uncontrolled fibroblast proliferation [23]. Our RNA sequancing exhibited that the development process of IUA was association with immune activation, endothelium development and epithelial cell development in early phase, while it presented high enrichment of cell-matrix adhesion and extracelular matrix organization in later phase. When the implantation of ADSCs/AAM was used to repair endothelial injury, RNA sequancing indicated that ADSCs could regulate the immune effetor process and neutrophil activation and endocytosis, which is beneficial to reduce the immune response. According to previous reports, ADSCs could reduce the expression level of pro-inflammatory cytokines, such as IL-1β, TNF-α and IL-8, and increase the expression of anti-inflammatory cytokines, such as IL-6 and IL-10 [24–25]. ADSCs may inhibit the overproduction of collagen by regulating inflammatory cytokines [26]. This study showed that the up-regulation of anti-inflammatory cytokines (IL-6, bFGF) and down-regulation of pro-inflammatory cytokines (IL-1 β, TNFα) at 3 and 7 days after ADSCs/AAM transplantation providing the best results when comparing with IUA or simple AAM transplantation group. However, at 14 days after surgery, there were no significant differences in all inflammatory factors between ADSCs/AAM and AAM group, indicating that ADSCs exerted its suppression of inflammatory response at an earlier stage. These phenomena partially suggest the mechanism of ADSCs on promoting endometrium regeneration in IUA model.

By the way, the particular strength of this study was the high-precision curettage procedure to the depth of the muscular layer, which was critical to evaluate the regenerative ability of ADSCs in IUA model. However, the potential long-term complication was still high abortion rate according to our pregnancy test. Although our study indicated better regenerated endometrium associated with ADSCs, it remained uncertain whether it would recover to normal endometrium. On the other hand, pregnancy test with larger amounts should be performed.

In conclusion, our outcomes demonstrated that AAM loaded with ADSCs could effectively prevent the formation of fibrosis and promote endometrium regeneration in rat IUA models. Although the exact mechanism of ADSCs in treating IUA remained unclear, our study suggests ADSCs regulated the inflammatory response by providing an anti-inflammatory environment and enhancing the regeneration of endometrium.

Method

Animals

96 female Sprague-Dawley (SD) rats weighted 220-240 g were purchased from Silaike Corporation (Shanghai, China). The protocol was approved by the Institutional Review Board and the Ethics Committee of our hospital. All animal experiments were approved by the Institutional Animal Care and Use Committee of Fudan University, Shanghai, China.

Culture, identification and multilineage differentiation of ADSCs

The rat inguinal subcutaneous fat was collected, chopped manually and digested with the same amount volume of PBS, supplemented with 0.075% type I collagenase (Washington Biochemical Corp, USA) with gentle shaping at 37 ℃ for 60 minutes. After neutralizing the enzyme, the digested tissue was centrifuged at 2000 g for 10 minutes and filtered with double-layer gauze to remove large pieces of debris. The pellets were resuspended in LG-MEM containing 10% FBS, 100 ug/ml streptomycin and 100 U/ml penicillin solution and plated in 100 mm culture dishes (Falcon, USA). After the fusion rate reached 70-80%, the cells were passaged to next generation for further experiment. ADSCs were identified via the cell surface antigens CD45, CD90, CD11, CD44, and CD29, using a flow cytometry assay. The multi-lineage differentiation potential of ADSCs was checked by adipogenic, osteogenic and chondrogenic differentiation assays at the fourth passage. Adipogenesis was induced by adipogenic induction medium (Gibco) for 14 days and confirmed by Oil red O staining to show intracellular lipid accumulation. Osteogenesis was induced by osteogenic induction medium (Gibco) for 21 days and calcium deposition was shown by Alizarin red staining. Chondrogenic differentiation was identified by toluidine blue staining by chondrogenic induction medium (Gibco) for 28 days.

Preparation of human AAM

Fresh human amniotic membrane (AM) was obtained from healthy patients under sterile conditions. The preparation of AAM was based on the report of Koizumi et al. Briefly, fresh AM was mechanically separated from the chorion membrane. After an entire wash with 0.9% sodium chloride solution for three times, AM was incubated with 0.02% ethylenediaminetetraacetic acid (EDTA) solution at 37 °C for 2 h, continuously stirring for acellularization. Then, the cellular debris was washed with PBS for three times to remove the remaining cellular debris.

The construction of ADSCs/AAM compounds

Before implantation, the AAM was cut into small pieces of 2 × 0.5 cm and soaked in normal saline for 10 min. ADSCs at passage 3 were resuspended in culture medium at a density of 5× 10⁵/ml. 100 ul cell suspension were evenly seeded in AAM to infiltrate the cell surface. After cells were incubated at 37 ℃ for 4 h to allow cells adhere to the membrane, the growth medium was added for further culture. After 24 h of culture, ADSCs/AAM constructs were transferred to new wells for subsequent culture in vitro.

Scanning electron microscopy (SEM)

Deposition of ADSCs on AAM was examined by scanning electron microscopy 1 week after implantation. Then, the samples were mounted, sputter-coated with gold, and viewed by SEM to observe adhesion and extracellular matrix (ECM) deposition under the surface of AAM.

Live/dead staining

The viability of ADSCs on AAM was evaluated at 3 and 7 days after implantation by a Live/Dead cell staining kit (Biovision, USA). Briefly, ADSCs/AAM constructs were washed with PBS and incubated in the assay reagents (2 uM calcein AM and 4 uM ethidium homodimer-1) for 15 min. Then, the stained living and dead cells were detected by confocal laser microscopy with a band-pass filter (FITC and rhodamine). Live cells emitted green fluoresce, and the nucleus of dead cells were dyed red.

Surgical procedure

The rat IUA model was established using the mechanical damage method [27]. Briefly, rats were anesthetized with 300 mg/kg 10% chloral hydrate and the abdominal cavity was open after iodophor disinfection. Then, slowly pick out and cut the uterus 2 cm from the upper one-third of the upper uterus. The endometrial tissue was then scraped to the depth of the muscular layer. In ADSCs/AAM group, the constructs were introduced to cover the damage area. In AAM group, single AAM was placed in the lesion of uterine. In IUA group, the lesion was directly sutured to heal itself. For sham operation group, the uterus was exposed to air for 20 min after opening the abdomen. At last, 6–0 absorbable suture was used to suture uterus intermittently. After washing the abdominal cavity with normal saline, the rectus fascia and skin were sutured with 4–0 silk suture. At day 3, 7, and 14 post-operation (n = 12 with 12 uteri for each time point), the whole uterus was dissected and sliced transversally for further evaluation.

Histologic examination and immunohistochemical staining

After the uterine tissue was fixed with 4% paraformaldehyde and embedded in paraffin, the samples were cut into 4-6 μm sections for H&E and Masson’s trichrome staining. The light microscope was used to obverse the morphological changes. Five fields in each image were selected to count. Image Pro-Plus 6.0 (IPP 6.0) was applied to analyze endometrial thickness, total number of endometrial glands, and fibrosis area. For immunohistochemistry staining, samples were performed to detect CD31, GB13063, 1:200, Servicebio, (an indicator of endothelial cells of microvessels), vimentin, ab92547, 1:200, abcam, (a marker of stromal cells), VEGF, ab32152, 1:200, abcam, (a vascular marker) and E-cadherin, sc-8426 1:50, Santa Cruz, (a marker of epithelial cell).

Next-generation RNA sequencing and bioinformaticsanalysis

Total RNA was extracted from the whole uterus in four groups at 3 and 14 days after surgery with TRIzol according to the manufacturer’s protocol (Invitrogen). The IIlumina standard kit was used according to the TruSeq RNA SamplePrep Guide (IIlumina). Magnetic beads containing oligo (dT) were used to isolate poly(A) mRNA from total RNA. Purified mRNA was then fragmented. Using these short fragments as templates, random hexamer primers and reverse transcriptase (SuperScript II, Invitrogen) were used to synthesize the first-strand complementary DNA (cDNA). The second-strand cDNAwas synthesized by using buffer, dNTPs, RNase H, and DNA polymerase I. Short double-stranded cDNA fragments were purified with QIAquick PCR extraction kit (Qiagen) and eluted with EB buffer for end repair and the addition of an “A” base. The short fragments were ligated to Illumina sequencing adaptors. DNA fragments with selected size were gel-purified with QIAquick PCR extraction kit (Qiagen) and amplified by PCR. The library was then sequenced on Illumina HiSeq™ 2000 sequencing machine. The library size was 400 bp, read length was 116 nt, and the sequencing strategy was paired-end sequencing. The clean reads were used for subsequent analysis and were mapped to the reference genome by TopHat. The gene expression was measured by the number of uniquely mapped fragments per kilobase of exon per million mapped fragments (FPKM). The R package limma was used to create differential expression genes. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) bioinformatics resource was used to annotate gene function and pathway.

RNA isolation and polymerase chain reactions

In orderto investigate the regulation effect of ADSCs on inflammatory response, real-time PCR was performed. According to the manufacturer’s protocol, RNAprep Micro Kit (TianGen Biotech, Beijing, China) was used to extract total RNA. Total RNA samples were extracted from excised uterine horns with RNAiso Plus (Takara Bio) and dissolved in water treated with diethyl pyrocarbonate. RNA concentrations were quantified using NanoDrop 2000 spectrophotometery (NanoDropTechnologies, 1f in of TGF-β and VEGF in ADsN Thermo Scientific). 2 ug total RNA was reverse-transcribed into cDNA in a 20 ul reverse transcription system with the Primestar extaq cDNA Synthesis Kit (TaKaRa). The reactions were performed and monitored in a T3 thermocycler (Biometra). Real time PCR was performed using a quantitative real time amplification system (MxPro-Mx3000P, Stratagene, La Jolla, CA). SybrGreen PCR MasterMix (Applied Bio-systems, Foster City, CA) was used in each reaction. To compare transcription levels of target genes in different quantities of sample, the quantified cDNA transcript level (cycle threshold) to that of GAPDH was used for normalization of real-time PCR results. Each sample was assayed three times.

Fertility test

The function of the scarred uterine horns was assessed by testing whether they were capable of receiving fertilized ova and supporting embryos to the late stage of pregnancy. At day 28 post-transplantation, another subset of rats (n = 6 with 12 uterine horns) from each group was mated with proven fertile male Sprague-Dawley rats. The rats were euthanized 14 days after the presence of vaginal plugs, and each uterine horn was examined for numbers, sizes and weights of fetuses, as well as sites of implantation.

Tracing of ADSCs in vivo

Dil is a nontoxic fluorescent marker of cell membranes, used to track for implanted cells. ADSCs were labeled with Dil (SigmaeAldrich, St Louis, MO). After washing, stained cells were cultured in sterile phosphate-buffered saline. Then, the Dil-labeled ADSCs were resuspended in the culture medium at a density of 5× 10⁵/ml and seeded on the AAM scaffold to form the ADSCs/AAM compound, which was implanted into the injured uterine. At day 3, 7, and 14 after implantation, the rats were sacrificed and uterine tissues were collected and frozen at −80 °C. The frozen tissues were continuously cut into 4-μm sections, and the nucleus was stained with DAPI. Then, the samples were observed by fluorescence microscopy (magnification × 100, Olympus, Tokyo, Japan).

Statistical analysis

All data are reported as means± standard deviation (SD) analyzed by SPSS 20.0. Statistical analysis was performed by Student’s t test for comparisons of different groups. A p value of less than 0.05 was considered statistically significant.

References

Di Spiezio Sardo A, Calagna G, Scognamiglio M, et al. Prevention of intrauterine post-surgical adhesions in hysteroscopy. A systematic review. European journal of obstetrics, gynecology, and reproductive biology. 2016;203:182–92.
March CM. Asherman's syndrome. Semin Reprod Med. 2011;29(2):83–94.
Guo EJ, Chung JPW, Poon LCY, Li TC. Reproductive outcomes after surgical treatment of asherman syndrome: A systematic review. Best practice & research Clinical obstetrics & gynaecology. 2019;59:98–114.
Robinson JK, Colimon LM, Isaacson KB. Postoperative adhesiolysis therapy for intrauterine adhesions (Asherman's syndrome). Fertility and sterility. 2008;90(2):409–14.
Salazar CA, Isaacson K, Morris S. A comprehensive review of Asherman's syndrome: causes, symptoms and treatment option. Curr Opin Obstet Gynecol. 2017;29(4):249–56.
Gholipourmalekabadi M, Sameni M, Radenkovic D, Mozafari M, Mossahebi-Mohammadi M, Seifalian A. Decellularized human amniotic membrane: how viable is it as a delivery system for human adipose tissue-derived stromal cells? Cell proliferation. 2016;49(1):115–21.
Hortensius RA, Harley BA. Naturally derived biomaterials for addressing inflammation in tissue regeneration. Exp Biol Med (Maywood). 2016;241(10):1015–24.
Farhadihosseinabadi BF, M, Tayebi T, Jafari A, Biniazan F, Modaresifar K, Moravvej H, et al. Amniotic membrane and its epithelial and mesenchymal stem cells as an appropriate source for skin tissue engineering and regenerative medicine. Artif Cells Nanomed Biotechnol. 2018;46(sup2):431–40.
Deans R, Abbott J. Review of intrauterine adhesions. J Minim Invasive Gynecol. 2010;17(5):555–69.
Maruyama T, Masuda H, Ono M, Kajitani T, Yoshimura Y. Human uterine stem/progenitor cells: their possible role in uterine physiology and pathology. Reproduction. 2010;140(1):11–22.
Nguyen HP, Sprung CN, Gargett CE. Differential expression of Wnt signaling molecules between pre- and postmenopausal endometrial epithelial cells suggests a population of putative epithelial stem/progenitor cells reside in the basalis layer. Endocrinology. 2012;153(6):2870–83.
Liu Y, Tal R, Pluchino N, Mamillapalli R, Taylor HS. Systemic administration of bone marrow-derived cells leads to better uterine engraftment than use of uterine-derived cells or local injection. Journal of cellular and molecular medicine. 2018;22(1):67–76.
Wang J, Ju B, Pan C, et al. Application of Bone Marrow-Derived Mesenchymal Stem Cells in the Treatment of Intrauterine Adhesions in Rats. Cellular physiology and biochemistry: international journal of experimental cellular physiology, biochemistry, and pharmacology. 2016;39(4):1553–60.
Shao X, Ai G, Wang L, et al. Adipose-derived stem cells transplantation improves endometrial injury repair. Zygote. 2019;27(6):1–8.
Panayotidis C, Weyers S, Bosteels J, van Herendael B. Intrauterine adhesions (IUA): has there been progress in understanding and treatment over the last 20 years? Gynecological Surgery. 2008;6(10):197–211.
Yang JH, Chen CD, Chen SU, Yang YS, Chen MJ. The influence of the location and extent of intrauterine adhesions on recurrence after hysteroscopic adhesiolysis. BJOG. 2016;123(4):618–23.
Salah RA, Mohamed IK, El-Badri N. Development of decellularized amniotic membrane as a bioscaffold for bone marrow-derived mesenchymal stem cells: ultrastructural study. J Mol Histol. 2018;49(3):289–301.
Azizi R, Aghebati-Maleki L, Nouri M, Marofi F, Negargar S, Yousefi M. Stem cell therapy in Asherman syndrome and thin endometrium: Stem cell- based therapy. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2018;102:333 – 43.
Gargett CE, Schwab KE, Deane JA. Endometrial stem/progenitor cells: the first 10 years. Hum Reprod Update. 2016;22(2):137–63.
Shamosi A, Mehrabani D, Azami M, et al. Differentiation of human endometrial stem cells into endothelial-like cells on gelatin/chitosan/bioglass nanofibrous scaffolds. Artif Cells Nanomed Biotechnol. 2017;45(1):163–73.
Zhao J, Zhang Q, Wang Y, Li Y. Uterine infusion with bone marrow mesenchymal stem cells improves endometrium thickness in a rat model of thin endometrium. Reproductive sciences. 2015;22(2):181–8.
Zheng SX, Wang J, Wang XL, Ali A, Wu LM, Liu YS. Feasibility analysis of treating severe intrauterine adhesions by transplanting menstrual blood-derived stem cells. International journal of molecular medicine. 2018;41(4):2201–12.
Bazoobandi S, Tanideh N, Rahmanifar F, et al. Induction of Asherman's Syndrome in Rabbit. J Reprod Infertil. 2016;17(1):10–6.
Kapur SK, Katz AJ. Review of the adipose derived stem cell secretome. Biochimie. 2013;95(12):2222–8.
Jahandideh S, Khatami S, Eslami Far A, Kadivar M. Anti-inflammatory effects of human embryonic stem cell-derived mesenchymal stem cells secretome preconditioned with diazoxide, trimetazidine and MG-132 on LPS-induced systemic inflammation mouse model. Artif Cells Nanomed Biotechnol. 2018;46(sup2):1178–87.
Sukho P, Cohen A, Hesselink JW, Kirpensteijn J, Verseijden F, Bastiaansen-Jenniskens YM. Adipose Tissue-Derived Stem Cell Sheet Application for Tissue Healing In Vivo: A Systematic Review. Tissue Eng Part B Rev. 2018;24(1):37–52.
Kuramoto G, Takagi S, Ishitani K, et al. Preventive effect of oral mucosal epithelial cell sheets on intrauterine adhesions. Human reproduction. 2015;30(2):406–16.

Regeneration of injured endometrium using acellularized amniotic membrane loaded with adipose- derived mesenchymal stem cells in a rat model

Abstract

Background

Aim

Methods

Results

Conclusion

Introduction

Result

Discussion

Method

Abbreviations

Declarations

References