1 Experimental materials
1.1 Experimental animals
Jinan Jinfeng Experimental Animal Co., Ltd. provided 24 4-month-old Japanese big ear rabbits, each weighing 2.5–3.5 kg. The rabbits were bred at the SPF Laboratory Animal Center of Affiliated Hospital of Jining Medical University [laboratory animal quality certificate No.: 37009700000659, license No.: SCXK (Shandong) 20180006]. All procedures of animal experiments conformed to the regulations of the Animal Ethics Committee of the Affiliated Hospital of Jining Medical University.
1.2 Reagents
RPMI 1640 medium, collagenase Ⅰ, fetal bovine serum (FBS), red blood cell lysis buffer (ACK), 1 × PBS, and trypsin were procured from Gibco (US).
1.3 Microcarrier 6 was provided by Elyon Bio-Technologies LLC (US).
2 Methods
2.1 Preparation of anal fistula rabbit model
For the preparation of the rabbit anal fistula model, referring to our previous study [10], sevoflurane was used for inhalation of general anesthesia and fixed after satisfactory anesthesia. Routine disinfection of perianal depilation was conducted, a sterile disposable cloth towel was laid, and an incision of the external anal fistula mouth from perianal to anal margin (0.5-1 cm) was performed with a self-made temperature control (shaping) electric knife. The internal anal fistula mouth is located in the anal canal 0.5–0.7 cm from the anal margin, forming a fistula between them, the temperature of the electric knife was controlled between 150–450 °C. After successful anal fistula preparation, a rubber band was implanted in the fistula. The two ends were ligated together with silk thread, and the rubber band could slide into the anal fistula. After 26 days, the rubber band was removed. Through visual inspection and probe examination of the perianal area of the rabbit anal fistula model, it was confirmed that the anal fistula model mimicked the human anal fistula model, and the model was deemed successful.
2.2 Extraction and culture of ASCs
After the rabbits received general anesthesia, the limbs were fixed, the skin of the operation area was treated, and normal disinfection was performed using iodophor, and then the sterile disposable cloth was laid. In the experimental group, a median incision about 4 cm long was made on the back of the rabbits, the subcutaneous tissue was separated, 5–20 g fat tissue was removed and placed into a sterile culture plate, and finally washed with normal saline. Excess hair and blood were removed, and cells were washed 2–3 times with PBS. The capsule, connective tissue, and blood vessels were removed under aseptic conditions. The adipose tissue was cut into 1 mm3 pieces with small scissors, digested using 5 ml 0.05% collagenaseⅠsolution in a constant temperature flask at 37 °C for 60 min, followed by the addition of RPMI medium containing 10% FBS to stop digestion. The suspension was centrifuged at 1000 rpm/min for 5 min to remove fat droplets in the upper layer and the supernatant. Cells were resuspended in PBS and passed through a 200-mesh filter. The supernatant was then centrifuged at 1000 rpm/min for 5 min and discarded. Cells were resuspended in RPMI medium containing 10% FBS, inoculated on 6-well plates, cultured in a 5% CO2 incubator at 37 °C, and the medium was changed every 3–4 days according to cell growth. The primary cells were cultured for two weeks and then digested with 0.25% trypsin. Cell morphology was observed using an inverted microscope. Photographs were taken and cells were collected after third generation passage.
2.3 Construction of the ASC-microcarrier 6 complex
The microcarrier-6 was soaked in 75% alcohol for 24 h and washed with PBS thrice. Next, the microcarrier 6 was incubated in RPMI 1640 medium for 24 h. Subsequently, the microcarrier-6 was modified with stromal cell-derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF); the concentration of both was 100 ng/mL, and the incubation time was 3 h. Cells in the log-phase growth stage were harvested. Using trypan blue, the percentage of living cells was found to be above 95%, and the cell concentration was adjusted to 2 × 107/mL. The ASC suspension was mixed with the modified microcarrier-6 and cultured in a 5% CO2 incubator at 370C for 24 h, until the cells reached a saturation state on the microcarrier-6 (under the microscope) [11].
2.4 Animal grouping
After successful development of the model, 14 rabbits were randomly divided into four groups: ASC-microcarrier 6 complex group (N = 4), simple ASC treatment group (N = 4), Operation group with thread hanging (N = 4), and control group (N = 2). Autologous ASCs were used for transplantation. For the ASC-microcarier-6 complex group, a mixture of 200 µl 2 × 107/ml ASC suspension and 100 µg microcarrier-6 was inoculated; the simple ASC treatment group was inoculated with 200 µl 2 × 107/ml ASC suspension; and the control group was inoculated with 200 µl 1 × PBS solution.
2.5 Surgical procedures for laboratory animals
(1) The inner mouth of the anal fistula was closed, we used an anal fistula scraper to remove necrotic tissue in the fistula and scrape the scar tissue and proliferative tissue of the inner wall of the fistula. Absorbable sutures were used to suture the inner mouth of the fistula. Physiological saline was injected from the outer mouth of the fistula to verify that the inner mouth was closed successfully. (2) ASC composite microcarrier scaffold transplantation was conducted by injecting 100 µl of tissue fluid with ASC composite microcarrier 6 scaffold into the tissue near the wound cavity of the fistula. To uniformly distribute cells, we used a multi-point injection method to inject the same amount of tissue fluid at 3, 6, 9, and 12 points around the fistula wound cavity. The non-invasive needle was replaced, and the remaining 100 µl of tissue fluid was injected through the external port into the entire fistula wound cavity. (3) The method of transplanting pure ASCs is the same as indicated in "2". (4) Operation group with thread hanging: the probe was slowly inserted from the outer opening of the fistula along the wound path of the fistula, the inner opening was penetrated and the probe end was bent, and finally pulled out from the anal opening. The thick wire connection of the tendon was tied to the probe head, and then the probe and the rubber band were pulled out. The rubber band was lifted to tighten the clamp along the base, and No. 7 silk thread was used under the vascular forceps. (5) Heavy rubber band ligation: The control group was inoculated with 200 µl of 1 × PBS. The experiment was ended by observing each feeding group daily and recording anal fistula healing.
2.6 Pathological examination and RNA sequencing
At the end of the experiment, the anal tissues (FIA-PBS, FIA-ETO, FIA ASCs, and FIA ASCs-Microcarrier 6) were fixed in 4% saline buffered formalin for pathological examination, and the same amount of tissues were frozen for RNA sequencing.
2.7 Statistical Analysis
The Student t-test was used to analyze the differences available in quantitative variables between the mentioned groups using the SPSS 16.0 software (SPSS, IL, USA). Herein, values of p < 0.05 were considered statistically significant.