Study population
A total of 22 patients in Nanjing Hospital of Chinese Medicine were recruited and enrolled in the study from January 2018 to October 2018. Their age ranged from 12 to 51 years old, with the average age being 28.86 ± 10.13 years old. Only one of them is female and they all signed informed consent before enrollment.
Inclusion criteria are as follows: ① diagnosis of complex Crohn’s fistula-in-ano, which meet the criteria of clinical practice guideline for the management of anorectal abscess, fistula-in-ano, and rectovaginal that was published by American Society of Colon and Rectal Surgeons in 2016. ② Patients with Crohn’s disease should control their disease in remission or mild active phase, that is, simplified Crohn’s Disease Activity Index (CDAI) is less than 6 points. ③ There is no evidence of cancer or precancerous lesions in enteroscopy 1 year before admission. ④ There is no other cardio-cerebrovascular diseases.
Exclusion criteria are as follows: ① acute infection stage of anal fistula (immature fistula). ② Patients with Crohn’s disease’s simplified CDAI > 6. ③ An autoimmune disease other than Crohn’s disease. ④ Patients with infectious diseases. ⑤ Patients who were allergic to anesthetics. ⑥ Patients who cannot tolerate liposuction. ⑦ Patients who were pregnant or were trying to become pregnant.
Study protocol
The study was conducted according to the principles of the Declaration of Helsinki. And the protocol was approved by the Ethics Review Committee of Nanjing Hospital of Chinese Medicine (Ethics Review No. KY2018011), and registered with the China Clinical Trials Registry (No. ChiCTR1800014599).
This study was designed as an open-label, randomized, controlled clinical trial to evaluate the efficacy and safety of ADSCs in the treatment of Crohn’s fistula-in-ano. The patients were divided into an observation group (ADSCs) and control group (incision-thread-drawing procedure). Patients in the control group received the traditional treatment of incision-thread-drawing procedure, while patients in the observation group received ADSC treatment. The specific treatment process is as follows: every patient was followed up for a minimum of 24 weeks to evaluate the efficacy and safety of ADSC treatment. Figure 1 shows the flow chart of the study.
Fistula preparation
After admission, patients received fistula preparation more than 2 weeks before ADSC injection, which included fistula exploration, curettage, and drainage with seton.
Preparation of ADSCs
Liposuction from the abdomen and thighs was performed on patients in the observation group (Fig. 2a, b). Then the fat was separated, cultured, proliferated, and identified as follows: First, the adipose tissue was washed with sterile normal saline, and then the corresponding concentration of collagenase I was added. After 60 min of shaking digestion at 37 °C, the upper lipid and liquid layers were centrifuged and absorbed, and the cells were re-suspended in normal saline and filtered by cell filters. The stromal vascular fraction (SVF) was obtained by removing the undigested tissue, centrifuging the filtrate, and discarding the supernatant. Trypan blue staining was used to count the number and activity of cells. SVF was then inoculated into culture flask. Serum-free medium and serum substitutes were added. The culture conditions were 5% CO2 and 37 °C. When the degree of cell fusion reached 70~80%, trypsin was added to digest the cells, and the digested cell suspension was collected and inoculated into the culture flask for subculture. ADSCs of the third or fourth generation were collected to detect cell morphology, viability, cell phenotype, endotoxin, bacteria, fungi, and mycoplasma (Figs. 3 and 4). After identification, it was frozen at − 80 °C, thawed and resuscitated on the day of injection, and transported to the operating room at 15–25 °C.
Injection procedure
More than 2 weeks after fistula preparation, patients in the observation group received injections with ADSCs. First, the fistula and its internal and external orifices were carefully explored with probes to avoid missing branches and pus cavities. After the exploration, the epithelial tissue of the fistula was destroyed from inside to outside by metal brush and electrocoagulation (COOK MEDICAL, USA, Registered Import of National Equipment 20153462399), and the necrotic tissue of the fistula wall was removed by washing with distilled water. After cleaning, the inner opening was closed with 2-0 vicryl. ADSC suspension containing 5 × 106cells/ml was injected uniformly into the inner orifice and around the fistula wall with a syringe (Fig. 2c). Multiple injections (> 4 times) were carried out in all quadrants. Finally, serum suspension containing 1 × 106 cells/ml was perfused into the fistula and the external opening was closed.
The dosage of ADSCs is based on the diameter and length of fistula measured before injection, and mainly according to the results of preoperative MRI and clinical evaluation at fistula preparation. The diameter of the fistula was less than 1 cm, and 1 ml ADSCs/cm was injected into the fistula. And 2 ml ADSCs/cm was injected into the fistula in the patients with the fistula diameter ranging from 1 and 2 cm.
Assessments
Evaluation of efficacy
The primary end point for efficacy was defined as the proportion of patients whose fistula had healed at months 3, 6, and 12 postoperatively. The researchers evaluated healing of the fistula through clinical evaluation at each follow-up (Fig. 2) and by magnetic resonance imaging (MRI) or endorectal ultrasonography (ERUS) at 3, 6, and 12 months postoperatively (Fig. 5). Healing was defined as the complete epithelialization of external openings (i.e., no pus outflow from the external openings under any circumstances) and no evidence of fistulas in MRI or ERUS.
The secondary end points for efficacy included simplified CDAI, Perianal Disease Activity Index (PDAI), Inflammatory Bowel Disease Questionnaire (IBDQ), pain scores with visual analog score (VAS), and Wexner score. And the patients completed these tests at each follow-up. In addition, we also recorded the inflammatory indexes of patients at baseline and month 3 postoperatively including high sensitivity C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and fecal calprotectin (FC).
Evaluation of safety
Safety was assessed by determining the incidence of adverse events (AEs) and serious AEs. During each follow-up, the AEs of patients were monitored. Perianal areas were assessed for the following: formation of abnormal tissue, persistence of or increased signs of inflammation, and any other observations that might indicate the impact of ADSCs.
Statistical analyses
The researchers used SPSS 22.0 software to process data. The last observation carried forward (LOCF) approach was applied in the case of missing data. Mean values were used to describe the measurement data, and t test or rank sum test were used for analysis. The counting data were described by frequency (constituent ratio) and analyzed by X2 test or Fisher exact test. P < 0.05 was considered statistically significant.