Study design and setting

The present randomized double-blind clinical trial was carried out in 2012 and 2013 to evaluate the efficacy of allogeneic transplantation of hADSCs for relieving fecal incontinence in patients with external anal sphincter defects presenting to Hazrate Rasoole Akram Hospital, Tehran, Iran. In this study, patients were consecutively entered and divided into two equal groups of sphincteroplasty with hADSC transplant (cell group) and sphincteroplasty with phosphate buffer saline (PBS; control group). The protocol of the study was approved by the Ethics Committee of Iran University of Medical Sciences. Researchers adhered to the principles of the Helsinki Declaration throughout the study and written informed consent was obtained from the patients. The protocol of the study was registered in the Iranian Registry of Clinical Trials (IRCT2016022826316N2).

Selection of patients

The present study was performed on 18 patients (15 women and 3 men, aged 25–78 years) who were randomly assigned to either the cell group (n = 9) or the control group (n = 9) (Fig. 1). Eligibility criteria are presented in Table 1. After taking their history and confirming fecal incontinence based on the Wexner score, [34] all subjects underwent 2D and 3D endorectal ultrasonography with a 360° probe (BK Pro Focus type 2202; OR, USA; Supplier: SafirMed) to assess sphincter damage.

Inclusion criteria	Exclusion criteria
− Age > 18 years − Wexner score ≥ 8 − Confirmation of external anal sphincter defect with endorectal ultrasonography − Patient consent for inclusion	− Pregnancy and breastfeeding − Participating in other trials within the past 30 days − History of artificial sphincter − Vaginal delivery within the past 6 months − Presence of chronic diseases − Allergy to bovine-derived materials − Autoimmune disease

A permuted block randomization (block size 3) was used to implement the random allocation sequence without stratification for baseline characteristics. An independent physician generated the random allocation sequence by a computer-based program, enrolled participants, and assigned participants to interventions. Four separate researchers prepared the suspensions, carried out the treatment administration, and recorded patients’ data. The patients and the surgeons who performed the surgery, sonographic, and physical examinations were blinded to the type of treatment. To ensure the blinding of the surgeon to the type of treatment, the cell suspension (6 × 10⁶ cells per 3 ml PBS) and placebo (3 ml PBS) were both prepared in packages which appeared to be the same.

Cell preparation

The isolation, preparation, and characterization of hADSCs, as well as corresponding results, were presented previously [35].

Isolation of hADSCs

Fat tissue was prepared from the superficial abdominal fat tissue layer in individuals aged 25–35 years who were referred to the general ward of Rasoul-e-Akram Hospital in Tehran, Iran for liposuction surgery after obtaining informed consent. The eligible patients had no history of positive HIV or hepatitis infections. Sampling was done in sterile conditions. The samples were transferred to the laboratory in a sterile plate containing DMEM/Hams F-12, FBS 10%, and penicillin/streptomycin (P/S) 5%. Isolation of stem cells was carried out based on a protocol described by Dubois et al. [36]. The fat samples were heated to 37 °C in a water bath prior to extraction. All of the extraction procedures were carried out under a sterile hood with sterile equipment. Washing the adipose tissue was performed in a tube containing P/S 1% solution (prepared by warming the PBS) and blood vessels and connective tissue were harvested to clear the tissue. The sample was transferred to a tube containing collagenase 0.1% and BSA 1.0% for tissue digestion. The tube containing the sample was then stored in a water bath for 30 minutes to complete digestion of the tissue samples. After tissue digestion, the tube containing the sample was centrifuged at a speed of 1200 rpm at room temperature for 5 minutes. After draining the supernatant fluid, the formed pellet was resuspended in a solution of BSA 1%, and centrifugation was carried out once again. To remove red blood cells, the formed pellet was resuspended with RBC lysis buffer and centrifuged. Finally, after washing with PBS, centrifugation, and evacuation of supernatant fluid, the reformed pellet was resuspended in DMEM/Hams F-12 culture medium containing FBS 10% and P/S 1% and was then transferred to a flask. Flasks were kept in the incubator (37 °C, CO₂ 5%, moisture 98%) until the fifth passage.

hADSC characterization

Flow cytometry was used to identify and confirm the hADSCs. After medium evacuation and washing with PBS, cells in flasks ready for the fifth passage were isolated from the flask by proximity to trypsin 25% for 4 minutes in the incubator. The cells were then used for flow cytometry after enzymatic neutralization, which was achieved by adding the same volume of medium and then transferring to a test tube. The cells were incubated with anti-CD45, anti-CD44 (conjugated to FITC), anti-CD90, anti-CD73, or anti-CD31 (conjugated to PE) for 30 minutes. All antibodies were purchased from BD Biosciences.

Surgical procedure

For the patients suffering from fecal incontinence due to sphincter damage, sphincteroplasty was performed. In this procedure, after opening the wound and excising the scar, the two ends of the muscle were exposed and washed with normal saline. The ends were then re-approximated without tension; 3-0 PDS sutures were utilized for repair. After washing with normal saline profusely and controlling any bleeding, 6 × 10⁶ hADSCs (3 × 10⁶ per 1.5 ml PBS into each end of the muscle) were injected slowly. The repair was then covered internally by an endorectal flap. Finally, outside the sphincter, excess skin tags were removed. The surgical procedure was completed without injection of antibiotics and antiseptics. Wound dressing was performed with gauze soaked in saline. In the following days, the wound was washed with abundant saline and fresh dressings applied. In the control group, all procedures were performed in the same manner but without implanting hADSCs.

In cases with external anal sphincter damage-related high trans-sphincteric fistula, it was usually impossible and inappropriate to primarily repair the sphincter because of the presence of extensions and collections around and above the fistula after laying them open. These wounds were left open and hADSCs (3 × 10⁶ per 1.5 ml in each end of the muscle) were slowly injected a few millimeters from the edge of the muscle.

Safety assessment

The present study evaluated the safety of hADSCs, which consisted of assessing the reported side effects after surgery until discharge from hospital and a 2-month follow-up of patients. Every 2 weeks, a full history was taken from the patients, and at the end of the second month, blood, urine, and stool analyses were performed. Safety of hADSCs was evaluated based on the WHO toxicity scale, in which the severity of adverse events is divided into four grades, namely mild, moderate, severe, and life-threatening [37]. Based on this scale, hematologic, chemistry, pancreatic and liver enzyme, stool, and urine analyses as well as cardiovascular, respiratory, digestive, neurologic, skin, and systemic (allergic reaction, headache, fever, and malaise) evaluations were done for all patients. Finally, mortality of the patients was also assessed.

Outcomes

The initial plan was to undertake a biopsy before final wound closure (after 8 weeks) and histopathological assessment of the repair zone for differentiation of muscle and fibrous tissue. However, due to the early closure of many wounds, obtaining a biopsy for histologic examination was not possible. Therefore, to check the results and the differentiation of fibrous tissue from muscle, sonography and electromyography (EMG) were used instead of histopathological examination.

Wexner score

To check muscle function after complete healing, the Wexner score was recorded by a single colorectal surgeon blinded to the study protocol.

Endorectal sonography

The mean of this ratio was finally recorded for each patient and also for each group. The first image was taken from the surface of the lesion, the second image from the mid part of the lesion, and the third image from the deepest part of the lesion.

Electromyography

After sonography and determination of the exact location of the gap, needle EMG was carried out by a single blinded physiologist. A 38-mm-long standard concentric EMG needle (38 mm × 0.45 mm; Umbo, Malaysia) was used. Standard filter settings (5 Hz–5 kHz), gain (200 μV/div, changed as needed), and sweep speed (10 ms/div) were used in the EMG system (Natus, Denmark). Subjects lay on their left side, with hips and knees flexed. Their right thighs were grounded electrically. The needle was inserted into the subcutaneous part of the external anal sphincter muscle to a depth of a few millimeters under the mucosa, about 1 cm from the anal orifice. For deeper parts of the external anal sphincter, needle insertions into the muscle were made at the anal orifice, at an angle of about 30° to the anal canal axis. Four quadrants of the external anal sphincter including the gap area marked by sonography were evaluated for spontaneous activity and voluntary motor unit action potentials (MUAPs). For evaluation of MUAPs, patients were asked to squeeze their sphincter, whilst for evaluation of spontaneous activity, patients had to relax the sphincter.

Statistical analysis

Sample size was determined based on evaluating the difference between recovery percentage in the control group and the hADSC treatment group. Based on the data of previous studies, the rate of recovery from fistula was reported to be 71% following hADSC transplantation and 12% in the control group [23]. Therefore, considering a 95% confidence interval (CI) (α = 0.05) and 80% power (β = 0.2), 11 patients in each group was enough for comparison.

Data were entered into SPSS 21.0 statistical software. Quantitative data were reported as mean and standard deviation (SD) and qualitative data were described as frequency and percentage. Because the data distribution was not normal, nonparametric analyses were used. To assess the difference between qualitative factors in the two groups, Fisher’s exact test was used. To compare age and the percentage of muscle in the injury site after intervention, the Mann–Whitney U test was applied. Because the Wexner score was different between the groups before intervention, to compare them after treatment an ANCOVA test (adjusted for the score before intervention) was used. In all analyses, p < 0.05 was considered significant.

Characteristics of hADSCs

Regarding cell culture, hADSCs were cultured in DMEM/F12 medium with FBS 10%. After the fifth passage, the cells were assessed using an inverted microscope equipped with a camera, which indicated spindle-shaped cells with fibroblast-like appendages (Fig. 2a).

In flow cytometry analysis, hADSCs had special surface markers of mesenchymal cells (positive for CD73, CD44, and CD90). At the fifth passage, a high percentage of cells expressed these markers as CD44 (75.77%), CD73 (97.40%), and CD90 (77.23%). Unlike these markers, hADSCs had no other markers including CD31 or CD45 (negative for these two markers). In fact, cells at the fifth passage expressed a very small percentage of CD31 (2.90%) and CD45 (3.37%) markers (Fig. 2b–d).

Baseline characteristics of patients

Adult patients with fecal incontinence were recruited for treatment by hADSCs or by placebo (3 ml PBS) from January 2012 to December 2013. Participants were assessed at the time of randomization (baseline) and followed up at 2 months.

Safety assessment

All of the hADSC-treated patients were evaluated for safety. No case of discontinuations due to side effects was detected. There were no cases of hemorrhage, need for blood transplants, pain, diarrhea, colon inflammation, infection, allergic reactions, recurrence of disease, readmission, and mortality. In addition, other side effects relating to hematologic, chemistry, pancreas and liver enzyme, stool, and urine analyses as well as cardiovascular, respiratory, digestive, neurologic, skin, and systemic (allergic reaction, headache, fever, and malaise) evaluations were absent in the patients. Only one case (11.11%) of erythema was seen in the site of surgery, which was closely followed; therefore it did not lead to any serious problems for the patient and was relieved completely.

Patients’ outcome

The mean baseline Wexner score of patients in the control and hADSC-treated groups was 6.0 ± 1.18 and 10.33 ± 0.87, respectively (p = 0.02). Two months after surgery, the Wexner scores of the control (p = 0.01) and hADSC-treated (p = 0.01) groups significantly decreased and reached 2.67 ± 0.62 and 6.44 ± 1.08, respectively. Based on nonparametric ANCOVA, the efficacy of conventional therapy and cell therapy was not significantly different (p = 0.36) (Table 2).

The results of endorectal sonography after complete wound healing and at least 8 weeks after surgery and injection of the cells clearly revealed the presence of scattered islands in the repair site compared with the control group (Fig. 3). Measuring the amount of muscle in the repair site using ImageJ/Fiji 1.46 software showed that the mean ratio of the area occupied by muscles was 18.85 ± 5.06% in the cell group and 11.65 ± 7.75% in the control group, which indicates a significant difference (p = 0.02) (Table 3).

Variable	Total	Control group	hADSC-treated group	p
Wexner score (mean ± SD)
Baseline	8.17 ± 3.74	6.0 ± 1.18	10.33 ± 0.87	0.02*
Post intervention	4.56 ± 3.22	2.67 ± 0.62	6.44 ± 1.08	0.36#
Standardized mean difference (95% confidence interval)a	3.73 (2.61–4.86)	3.63 (2.0–5.07)	3.97 (2.31–5.62)	0.41
Percentage of muscle occupied area (mean ± SD)	15.25 ± 7.34	11.65 ± 7.74	18.85 ± 5.06	0.02*
Electromyography recording (n, %)
Negative	10 (58.82)	9 (100.0)	1 (12.50)	0.002*
Positive	7 (41.18)	0 (0.0)	7 (87.50)

Control group sphincteroplasty alone, hADSC-treated group human adipose-derived stem cells + sphincteroplasty, SD standard deviation

^aBased on Hedge’s g

*Based on Mann–Whitney U test

^#Based on nonparametric ANCOVA test

EMG in the injected cell group showed that five out of nine views had normal action potentials. One of the cases had no action potential that was consistent with sonography results indicating the lack of muscular islands in the lesion site after injection of cells. In another case, the results of EMG were not recorded due to omission of the test. In one patient, the sphincter had been cut at two sites and a normal action potential was revealed in one site, but not in the other. In another patient, a normal action potential was subsequently recorded at one site and both neurogenic and spontaneous activities were recorded at the other. EMG recordings were negative in all control patients (Table 3 and Fig. 3). Fisher’s exact test revealed that EMG activity was significantly higher in hADSC-treated compared with control patients (p = 0.002). No adverse event was seen at the 2-month follow-up.