Experimental animals
All New Zealand female rabbits were purchased from Xi’an Bioscience Co. Ltd. (China), and housed separately in wire cages at room temperature (about 25℃) in 12:12 h light/dark cycle, fed adlibitum with free water access. All animal handling procedures were carried out according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH publication no. 85–23, revised 1996). The Ethics Committee of Ningxia Medical University approved experimental protocols in this study. The rabbits with an age of 3 weeks or 0.8 kg were used as donors for BMSC isolation and characterization. Animals with weights of 2.0-2.5 kg were employed for experimental controls or treatments.
Isolation and characterization of BMSCs
BMSCs were isolated from the tibia and femur of 3-week-old rabbits using the method of adherent culture of whole bone marrow. Briefly, under complete aseptic conditions, bone marrow cells were harvested by flushing the marrow cavity with low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) [Gibco, Gainesville, MD, USA]. The suspended cells were then collected by centrifugation at 1500 rpm for 5 min. The isolated cells were cultured in DMEM (Gibco, Cat#:1655697) containing 10% fetal bovine serum, 1% penicillin-streptomycin (Gibco, Cat#:1631430) at 37℃ in a humidified atmosphere with 5% CO2. The culture medium was refreshed every 2–3 days until the confluence reached 90%. The cells of the 3rd passage were used for the in-vitro test and transplantation. BMSCs were identified by specific expression of CD44 (Bioss, bs-4916R), CD29 (ab78502, Abcam), CD90 (Bioss, bs-0778R), and CD45 (ab30446, Abcam) on BriCyte E6 (Mindray DS US Inc., NJ, USA) flow cytometer. Adipocyte induction of BMSCs: when the cells reached 100% confluence, complete medium was replaced with adipogenic induction medium in the next 3 weeks. Then, Oil-Red-O staining was performed. Osteoblast induction of BMSCs: when the cell reached 80 to 90% confluence, complete medium was replaced with osteogenic induction medium in the next 3 to 4 weeks. Then, Alizarin-Red staining was performed.
Labeling BMSCs with PKH26 fluorescent dye
To track the transplanted cells in vivo, the BMSCs were harvested during the 3rd passage and labeled with PKH26, a lipophilic red fluorescent linker dye (Cat. # MINI26, Sigma Aldrich, St. Louis, MO, USA). Briefly, the BMSCs were centrifuged and washed 2 times in serum-free medium and pelleted. Then, 1ml Diluent C and 4ul PKH26 reagent was added, mixed using a pipette, and incubated for 5 min at room temperature. Subsequently, 2 mL of FBS was added to stop the staining. After centrifugation, the supernatant was discarded and cells were resuspended in fresh medium. The PKH26-labeled BMSCs were examined using phase-contrast Olympus CKX53 fluorescent microscope (Olympus, Tokyo, Japan) at 48 hours post the staining. The cells were maintained in the growth medium prior to being injected intravenously into the uterine cavity of rabbits. After retrieval of the uteri on the 3 d, 5d, and 7 d after BMSCs injected, they were embedded in OCT and sectioned (5-µm thick). Representative sections were stained with DAPI to examine the survival and migration of BMSCs in vivo.
Generation of rabbit IUA model
The IUA animal model was generated by a dual damage method of mechanical curettage and lipopolysaccharide (LPS, 6 mg/L, Sigma) infection. Briefly, rabbits were exposure to general anaesthesia after administration of 10% chloral hydrate (3ml/kg) by intravenous injection, and the abdominal wall and cavity was surgically opened to expose the bilateral uterus under sterile conditions. A 2-mm transverse incision in bilateral the uterus was prepared at about 1.5 cm from the junction of the bilateral uterus with ophthalmic scissors, a miniature endometrial scraping spoon was inserted into 2/3 of the uterus, and the scraping spoon was repeatedly rotated until the uterine wall appeared rough and grainy. LPS-soaked cotton thread was then placed into the uterine cavity through the uterine incision, leaving about 5 cm of cotton thread outside to remove the cotton thread after 48 hours. Finally, the abdominal cavity and wall were closed in layers, and 80,000 U/100 mg penicillin (North China Pharmaceutical Co., Ltd) was intramuscularly injected to prevent infection 5 days after the operation. One week after modeling, animals were utilized for experiments. The study comprised of 75 rabbits, which were randomly assigned to the following 5 groups (n = 15 for each group): sham operation group (sham), IUA model group (IUA), E2 treatment group (IUA + E2), BMSCs treatment group(IUA + BMSCs), and BMSCs combined with E2 treatment group (IUA + BMSC + E2 ). The rabbits in the sham operation group had laparotomy without any treatment, in the estrogen groups received intramuscular injections of estrogen (0.1 mg/ kg) for 21 days, in the BMSCs group were injected with 150 µl BMSC suspension in the injured uterine segment, while the IUA model group received PBS as a control. Five rabbits were euthanized at time 7 d, 14 d and 21 d and bilateral uterus were obtained at each time points for the histopathological and immunoreactive evaluations and molecular study.
H&E and Masson staining
The endometrial tissue samples were fixed with 4% paraformaldehyde (Sigma-Aldrich, Cat# P6148) for 24 h and then embedded into paraffin blocks after dehydration and hyalinization. The paraffin-embedded tissues were cut into 4-um-thick slices for hematoxylin and eosin (H&E) histochemical staining to evaluate the alterations of endometrial morphology. Five high-magnification fields were evaluated for each HE-stained section, and the number of glands in each field of view was counted and averaged. The slices were also stained by Masson method. To quantitatively evaluate the degree of endometrial fibrosis, five random fields of each Masson-stained slide were chosen to calculate the ratios of endometrial fibrotic areas/total endometrial areas (excluding the uterine cavity) using IPP 6.0. The ratios averaged per group were obtained.
Immunohistochemical staining
Samples were fixed in 4% paraformaldehyde and embedded in paraffin. The transverse paraffin sections were deparaffinized using xylene, rehydrated through a series of alcohol gradients (100–70%) and rinsed in water. Then, the sections were incubated in 3% hydrogen peroxide for 30 min to inactivate endogenous peroxidase and incubated with the following primary antibodies: anti- CK7 (Abcam, USA, 1:500), anti-vimentin (Abcam, USA, 1:400), anti-p-Keratin (Abcam, USA, 1:200), anti-a-SMA (Abcam, USA, 1:200) at 37℃ for 2 h. After washing, the sections were incubated with the biotinylated secondary antibody for 1 h at room temperature. Protein expression was visualized with diaminobenzidine (Dako Cytomation, Carpinteria, CA, USA) staining. The number of positively stained cells and absorbance were quantified at five randomly selected fields per section.
Immunofluorescence staining
Uterus of rabbit was frozen after embedding in OCT compound (Tissue-Tek; Sakura). Frozen blocks were cut into 6 µm in thickness with a microtome at − 20°C and dried > 2 hours at room temperature. Sections were fixed in cold acetone for 15 minutes and kept at − 80°C. Sections were incubated with blocking solution consisting of 5% donkey serum and 0.3% Triton-X 100, followed by incubation with antibodies against CK7 (Abcam) and Vimentin (Abcam) at 4°C overnight. After the tissue was washed with PBS solution 3 times, the slices were incubated with fluorochrome-conjugated antibodies (Invitrogen) at room temperature for 2 hours. Then, the slices were washed with PBS 3 times before being incubated with Fluoroshield Mounting Medium with DAPI (Abcam). Images were taken with a fluorescence microscope (model BX-61, Olympus).
Immunoblotting assay
The total protein was extracted, and the protein concentration was determined by BCA method. Total protein was separated on a 10% SDS-PAGE gel, and then transferred to PVDF membranes. The membranes were blocked with 5% defatted milk for 1 h at room temperature and incubated with specific primary antibodies overnight. Membranes were washed three or four times with PBS and then incubated with the secondary Antibody at 37℃ for 1 h in the dark. Finally, the proteins of interest were visualized using ECL in the BioImaging System (BIO-RAD, USA). GAPDH was used as an internal control to normalize the relative expression of each protein of interest. Three replicates were performed in each group.
Statistical analysis
Statistical analysis was performed using SPSS 20.0 (IBM, USA) and GraphPad Prism version 6.0 (USA). The data are reported as means ± SD. Two samples were compared using independent sample two-tailed t-test. Statistical differences among multiple groups were determined by one-way analysis of variance (ANOVA). P<0.05 was considered to be statistically significant.