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.
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.
The rat IUA model was established using the mechanical damage method . 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.
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).
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.