Ethical approval statements
This investigation was performed in accordance with relevant guidelines and regulations of animal studies and all experimental protocols were approved by ethical committee of Bushehr University of Medical Sciences (Permission number: IR.BPUMS.REC.1399.084).
Thirteen female (1 donor and 12 recipients) adult healthy New Zealand white albino rabbits (Oryctolagus cuniculus) weighing 3500–4000 g were purchased from and housed in the Center of Comparative and Experimental Medicine, Shiraz University of Medical Sciences. They were maintained singly in stainless steel cages under the appropriate condition (20 ± 2 °C temperature, humidity 60%, and 12-h light/dark cycle) and had free access to food and water. Their food was supplemented by adding carrot and parsley.
To establish the BM-MSCs culture, under sterile condition both femur and tibia from a rabbit were excised and carefully cleaned of adherent soft tissue. The ends of the bones were cut away and bone marrow was harvested by flushing with 10 mL syringe with Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin (Sigma-Aldrich) and 1% L-glutamine (Sigma-Aldrich). After washing and centrifugation at 1200 rpm for 5 min, cell pellet was re-suspended in fresh medium and cultured in a 75-cm2 flask in the supplemented DMEM medium. The flask was incubated in a CO2 incubator (5% CO2, 37 °C, and saturated humidity). The first culture media was changed after 24 h to remove non-adherent cells.
The adherent cells were cultured till 80–90% confluence by exchanging the subsequent medium every 3–4 days and then passaged to expand the MSCs population. The adherent cells were washed with phosphate buffer saline (PBS) and the cells were harvested after for 2–3 min treating with 0.25% trypsin (Gibco) and the enzyme was inactivated with same amount of culture medium. Spindle-shaped morphology of BM-MSCs was observed using light microscopy at every passage. The cells were sub-cultured two times to obtain a sufficient number for evaluation of stemness characters and cell therapy. Cells in the second passage were collected and counted using a hemocytometer.
They were cryopreserved through the conventional method by dimethyl sulfoxide (DMSO; MP Bio, France) and were aliquoted into sterile cryovials at a density of 2 × 106 viable cells/ml. Before cell characterization or cell therapy, the frozen cryovials were quickly thawed in a 37 °C water bath. Before the ice clump completely thawed, 1 mL of supplemented DMEM medium was added. After re-suspension of the cells in the fresh medium, they were cultured and subcultured just one time in the same condition and medium as has been explained above. Except cell morphology and plastic adherent characters of isolated cells, to confirm that the isolated cells were MSCs, the potential of differentiation to adipocytes and osteoblasts as well as their surface markers were detected.
Reverse transcription-polymerase chain reaction (RT-PCR)
BM-MSCs were examined for expression of surface markers using RT-PCR. Total RNA of BM-MSCs at passage 3 was extracted according to manufacturer’s instructions using column RNA isolation kit (Denazist-Asia, Iran). Total RNA concentration was determined by nanodrop spectrophotometery. Before reverse transcription, the RNA samples were digested with DNase to remove contaminating genomic DNA. After that, complementary DNA (cDNA) synthesis from DNA-free RNA (500 ng) samples was done by using Accu Power Cycle Script RT PreMix Kit (Bioneer, Korea) according to the manufacturer’s protocol.
Specific primers were designed based on sequences corresponding to highly conserved regions of CD34, CD45, and CD73 in rabbit.The primer sequences used are summarized in Table 1. The micro-tubes containing requirements of PCR reaction up in a 20 µL mixture were transferred to a Thermocycler (Eppendorf Mastercycler Gradient, Eppendorf, Hamburg, Germany). The RT-PCR amplification conditions for the surface markers were performed in 30 cycles of amplification including denaturation at 95 °C for 30 sec, annealing at 64 °C for 30 sec, and extension at 72 °C for 30 sec, with deployment 2 min at 95 °C for primary denaturation and 5 min at 72 °C for final extension. PCR products were run on 1.5% agarose gel electrophoresis and visualized by UV light (UVtec, Cambridge, UK).
Sequences of RT-PCR primers used to quantify the expression of bone marrow-derived mesenchymal stem cells specific surface markers (CD73) and hematopoietic stem cells specific surface markers (CD45 and CD34) in rabbit
Amplicon length (bp)
Osteogenic and adipogenic differentiation assay
In order to evaluate the differentiation potential of BM-MSCs, cells of passage 3 were used and osteogenic and adipogenic differentiation were induced. For osteogenic differentiation BM-MSCs were seeded in a 6-well plate. After the cells reaching 70% confluence, they were cultured for 3 weeks in osteogenic medium containing low glucose DMEM supplement with 100 nM dexamethasone (Sigma-Aldrich), 0.05 mM ascorbate-2-phosphate (Wako Chemicals, Richmond, VA, USA), 10 mM b-glycerophosphate (Sigma-Aldrich), 1% antibiotic/antimycotic and 10% FBS. The half of medium was replaced every 3 days. At day 21, the cells were fixed by 10% formalin solution (Sigma-Aldrich), and then stained using Alizarin red (Sigma-Aldrich) to detect calcified extracellular matrix and osteogenic differentiation.
For adipogenic differentiation BM-MSCs were seeded in a 6-well plate. When they reached 70% confluency, were induced to adipogenic differentiation with adipogenic induction medium containing DMEM low glucose, 10% FBS, 0.5 mM isobutyl-methylxanthine (Sigma-Aldrich), 10% FBS, 0.5 mM isobutyl-methylxanthine (Sigma-Aldrich), 1 µM dexamethasone, 10 µM insulin, 200 µM indomethacin (Sigma-Aldrich). The plates were maintained for three weeks and medium was replaced every 3–4 days. At the end of period, the cultures were fixed by 10% formalin solution for 10 minutes. Fixed cells were subjected to oil red O (Sigma-Aldrich), which specifically stains lipid droplets.
Cell counting, growth curve and calculation of population doubling time (PDT)
Growth curves were plotted for BM-MSCs derived from rabbit bone marrow in order to evaluate growth kinetics of the cells. For the assessment of growth characteristics, BM-MSCs at passage 3 were seeded in a 24-well plate at a density of approximately 7.5 × 104 cells per well in triplicate. Cells were collected from each well 1–7 days after seeding and counted microscopically to draw a cell growth curve. The curve was drawn using GraphPad Prism (Version 5.01; GraphPad software Inc., San Diego, CA, USA).
To evaluate the in-vitro proliferation rate, the PDT value was determined for each studied cells. PDT was calculated using the formula PDT = T ln 2/ln (Xe/Xb), in which T is the incubation time in hours, Xb represents the cell number at the beginning of the incubation time and Xe corresponds to the cell number at the end of incubation time.
Preparation of BM-MSC-CM
The MSC-CM was collected from the third passages of cultivated BM-MSCs. In order to obtain the CM, BM-MSCs at passage three were cultured at a density of 106 cells per T75 flask. At 80 to 90% confluence, the BM-MSCs were washed three times with PBS and the media were replaced with 10 mL of FBS-free DMEM. After 48 h incubation, the media were collected and filtrated through a 0.2-µm filter to remove cellular debris and stored at -80 °C until use.
For induction of the model and treatment, double uteri of each 12 female rabbits were randomly divided into four groups of intact negative control, curettage positive control, BM-MSCs injection, and BM-MSC-CM injection in the way that two corresponding uteri from a rabbit were assigned in the different groups (n = 6). Except intact negative uteri, the caudal part of the other uteri was submitted traumatic endometrial curettage.
Briefly, the rabbits were anesthetized with a single intramuscular dose of a combination of ketamine 10% (35–40 mg/kg, Alfasan, Netherlands), and xylazine 2% (3–5 mg/kg, Alfasan, Netherlands) after pre-operative overnight fasting. Following midline incision as laparotomy, 5 cm long incisions were performed on the uteri. Incisions were located in the middle of each uterine tube. Using a scalpel blade through these incisions, the inside out everted endometrium was scratched (Fig. 1A). Whole thickness of endometrium was removed and it was continued until observation of bleeding as an indicator of curettage completion. Then uteri wall was sutured with 4 − 0 vicryl. The curettage positive control uteri were curettaged but not injected. Immediately, after suturing of uterus in both treatment groups, 1 mL of the BM-MSCs (2 × 106 cells) or BM-MSC-CM were injected into the uterus (Fig. 1B). The intact negative control uteri were not curettaged or injected. Using 3 − 0 vicryl, abdominal muscles were sutured and by 2 − 0 silk sutures, the skin was closed. At the end of operation, rabbits received flunixin meglumine (0.2 mg/kg, IM, Caspian–Tamin™, Iran) immediately and then every 24 hours in 3 doses. Penstrep-400 (Nasr™, Iran, IM) was injected as an antimicrobial, just after the operation and continued for 3 days.
Histomorphometry of uterus
Effect of both treatment techniques on regeneration of endometrium evaluated 30 days after treatment by histomorphometric comparing with the controls. Rabbits were sacrificed with a high dose of pentobarbitone (1 g, Specia, France). Both uteri of rabbits were fixed for two weeks in 10% formalin buffer. After fixation, segments were embedded in paraffin, and 5 µm thickness sections were made from each block in the area of curettage with the help of suture landmark.
They were stained with Masson trichrome to analyze histopathologic damages following curettage and the evolution of the regeneration process after treatment by evaluating morphologic indices and accumulation of fibrotic tissues in the curettage site. The slides were visualized and photographed using a light microscope (CX21, Olympus, Japan) equipped with an adjusted digital camera (AM423U Eyepiece Camera, Dino-Eye, Taiwan). On the transverse sections, endometrial area, lumen area, and total area of the uterus were measured using Dinocapture 2.0 software (Dino-Eye, Taiwan). The proportion of intact, damaged and regenerated endometrial luminal epithelium were estimated by calculation of different indices. Histomorphometric indices according to the previously described method (13) include lumen area/total horn area ratio (LA/THA), endometrial area/total horn area ratio (EA/THA), myometrial and perimetrial area/total horn area ratio (MPA/THA), endometrial area/uterine wall area ratio (EA/UWA), and myometrial and perimetrial area/uterine wall area ratio (MPA/UWA). Synechiae occurring, inflammatory elements and histopathologic changes were evaluated in intact, curettage and treatment uteri.
Image analysis of fibrosis
Three separate fields (200 × 200 µm2) of endometrial layer in 1 slides (n = 6) from different groups were cropped. Then, using color threshold of ImageJ, the green and blue pixels which showed the presence of collagen fibers were selected and percent of area of collagen were measured in each field.
Normality test of the histomorphometric ratio indices were carried out by Kolmogorov-Smirnov test. The mean and standard error (SE) ratios of LA/THA, EA/THA, MPA/THA, EA/UWA and MPA/THA were subjected to Kolmogorov-Smirnov normal test and data were analyzed by two-way ANOVA and LSD tests. Data were analyzed by SPSS 22 for windows (IBM SPSS Statistics for Windows, version 22, IBM Inc., Chicago, Illinois). P-value less than 0.05 was considered statistically significant. Means and SE are reported in charts (GraphPad Prism version 5.01 for Windows, GraphPad Inc. Inc., San Diego, CA, USA).