The Ecacy and Mechanism of Autologous Fat Stem Cells Combined with Microcarrier 6 Transplantation for Anal Fistula Treatment

Background: Adipose-derived stem cells (ASCs) function in multi-directional differentiation, proliferation, and tissue regeneration. It is not clear whether microcarrier 6 can promote the migration, differentiation, and regeneration of fat stem cells, and improve therapeutic effects for the treatment of anal stula. Methods: Japanese big ear rabbits were employed to establish an anal stula model, and ASC-microcarrier 6 mixed transplantation was used as a treatment. HE staining, immunohistochemistry, and RNA sequencing were used to observe the effect of ASC-microcarrier 6 transplantation on anal stula healing, in comparison with the ASC treatment group, rubber band operation group, and control group. Results: HE staining indicated scattered striated muscle cells and epithelial tissues in stula tissues of the ASC-microcarrier 6 complex group and the ASC treatment group, while a small number of lymphocytes were clustered around the microcarrier 6, and fat cell aggregation was seen in the ASC treatment group. RNA sequence analysis showed that differential genes were mainly concentrated in striated muscle cells, vascular smooth muscle, and other tissues. PI3K/AKT signaling pathway molecules were signicantly enriched, granulation tissue and lymphocyte inltration were observed in the rubber band string operation group, and a large amount of necrotic tissue was seen in the control group. Conclusion: Microcarrier 6 is benecial to the multi-directional differentiation of ASCs, which can provide a good environment for the survival of ASCs and promote stula healing.


Introduction
Anal stula is an infectious canal disease involving the rectum, anal canal, and perianal skin, and usually presents as a recurrent perianal infection. Anal stula cannot heal spontaneously, and surgery is the main treatment for anal stula at present [1]. However, conventional surgery is usually associated with a large wound surface and can damage the sphincter ani to varying degrees. Furthermore, surgery-related complications can lead to postoperative copracrasia, which can greatly affect the patients' life quality [2].
Developing alternative therapies to increase the healing rate of anal stula and maximally preserve anal function has long been an urgent need [3].
Adipose-derived stem cells (ASCs) are mesenchymal stem cells that are found in adipose tissues. They possess self-regeneration and multi-differential potential and belong to the class of adult stem cells [4].
As compared to the bone marrow-derived hematopoietic stem cells, ASCs have a shorter doubling time, faster doubling speed, and display the potential of trans-germ-layer differentiation [5]. Besides, ASCs provide several advantages, such as ease of availability, abundance in the human body, and good tissue regeneration and repair effects. Under different induction conditions, ASCs can secrete a variety of growth factors through autocrine and paracrine effects. Moreover, ASCs can be induced to differentiate into many cell types, thus promoting epithelial and vascular regeneration and local blood supply reconstruction and recovery. ASCs are ideal seed cells for tissue regeneration. ASCs are widely used in tissue repair, reconstruction, and in the prevention and treatment of diseases of several systems, including autoimmunity [6]. Clinical studies have reported the application of ASCs in the treatment of anal stula, which has been widely considered due to several advantages, such as small wound areas, no damage to the anal sphincter, and rapid postoperative recovery [7]. However, the e cacy and safety of ASCs are not clear.
Many researchers believe that in the early stage of transplantation (before new blood vessels have been completely formed), the transplanted ASCs require a constant supply of nutrients from the extracellular matrix and interstitial uid in the recipient tissues for their survival [8]. This explains the uncertainty regarding the success rate of ASC transplantation. Development of tissue engineering provides a new pathway to solve this problem. Microcarrier 6, manufactured by ELYON BIOTECHNOLOGIES LLC (USA), is composed of positively chargeable organic polymer composite and presents a multi-layer porous structure. Its pore size, positive charge density on its surface, and carrier particle size can be regulated by chemical synthesis. Microcarrier 6 is composed of organic compounds and displays resistance to contamination, low immunogenicity, good biocompatibility, and can be easily used in metabolic processes [9]. Our studies have shown that Microcarrier 6 complexed with cells provides a 3D multichannel scaffold structure for the cells, so that the transplant can quickly establish a connection with recipient tissues. With an independent and organic relationship formed between Microcarrier 6 and the cells, a stable growth microenvironment can form for transplanted cells, thereby improving treatment effects [9].
According to the existing research, ASCs have been shown to play a positive role in the treatment of anal stula, however, the e cacy requires further improvement. Microcarrier 6 can improve the survival rate and biological function of transplanted cells. ASCs and microcarrier-6 are used to repair and treat anal stula. The therapeutic effect of ASCs combined with microcarrier 6 is observed and its mechanism is discussed.

Preparation of anal stula rabbit model
For the preparation of the rabbit anal stula model, referring to our previous study [10], sevo urane was used for inhalation of general anesthesia and xed after satisfactory anesthesia. Routine disinfection of perianal depilation was conducted, a sterile disposable cloth towel was laid, and an incision of the external anal stula mouth from perianal to anal margin (0.5-1 cm) was performed with a self-made temperature control (shaping) electric knife. The internal anal stula mouth is located in the anal canal 0.5-0.7 cm from the anal margin, forming a stula between them, the temperature of the electric knife was controlled between 150-450 °C. After successful anal stula preparation, a rubber band was implanted in the stula. The two ends were ligated together with silk thread, and the rubber band could slide into the anal stula. After 26 days, the rubber band was removed. Through visual inspection and probe examination of the perianal area of the rabbit anal stula model, it was con rmed that the anal stula model mimicked the human anal stula model, and the model was deemed successful.

Extraction and culture of ASCs
After the rabbits received general anesthesia, the limbs were xed, 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 nally 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 mm 3 pieces with small scissors, digested using 5 ml 0.05% collagenase solution in a constant temperature ask 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 lter. 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% CO 2 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.

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 modi ed 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 × 10 7 /mL. The ASC suspension was mixed with the modi ed microcarrier-6 and cultured in a 5% CO 2 incubator at 37 0 C for 24 h, until the cells reached a saturation state on the microcarrier-6 (under the microscope) [11].

Animal grouping
After successful development of the model, 14 rabbits were randomly divided into four groups: ASCmicrocarrier 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 ASCmicrocarier-6 complex group, a mixture of 200 µl 2 × 10 7 /ml ASC suspension and 100 µg microcarrier-6 was inoculated; the simple ASC treatment group was inoculated with 200 µl 2 × 10 7 /ml ASC suspension; and the control group was inoculated with 200 µl 1 × PBS solution.

Surgical procedures for laboratory animals
(1) The inner mouth of the anal stula was closed, we used an anal stula scraper to remove necrotic tissue in the stula and scrape the scar tissue and proliferative tissue of the inner wall of the stula.
Absorbable sutures were used to suture the inner mouth of the stula. Physiological saline was injected from the outer mouth of the stula to verify that the inner mouth was closed successfully. (2) ASC composite microcarrier scaffold transplantation was conducted by injecting 100 µl of tissue uid with ASC composite microcarrier 6 scaffold into the tissue near the wound cavity of the stula. To uniformly distribute cells, we used a multi-point injection method to inject the same amount of tissue uid at 3, 6, 9, and 12 points around the stula wound cavity. The non-invasive needle was replaced, and the remaining 100 µl of tissue uid was injected through the external port into the entire stula 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 stula along the wound path of the stula, the inner opening was penetrated and the probe end was bent, and nally 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 stula healing.

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 xed in 4% saline buffered formalin for pathological examination, and the same amount of tissues were frozen for RNA sequencing.

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 signi cant.

1.Morphological observation of ASCs
After the primary cells were cultured for 1 day, it was found that some cells were round with visible nuclei that adhered to the walls of the culture ask. On the 2nd day, the number of adherent cells increased, and some adherent cells were deformed, with an increase in cell diameter. On the 3rd day, the cells gradually elongated resembling a polygon shape. On the 7th day, the adherent ASCs presented with a uniform morphology and a long spindle shape. On the 12th day, most adherent cells were fused, presenting a colony-like distribution and sh swarm-like arrangement (Fig. 1a). Lipid droplets could be seen in certain individual cells. After two weeks of culture, the cells were uniformly distributed, and most appeared long and spindle-shaped, with nuclei in the middle. After cell passage, the cells became quickly adherent to the walls with relatively uniform morphology and few heteromorphic cells. The cells presented with a colonylike or sh swarm-like arrangement pattern (Fig. 1b).
After digestion with trypsin, ASCs became a single-cell suspension and co-incubated with microcarrier 6 for 4 h. It was then found that the ASCs adhered well to the microcarrier 6 with uniform distribution, and reached a saturation state (Fig. 1c).

Observation of treatment effect in anal stula
After initiating anal stula treatment, the healing procedure was observed on a daily basis, for a total of three weeks. All animals survived during the surgery and follow-up period (Table 1). Table 1 Observation of treatment effect in anal stula. Compared with the thread-drawing procedure group, the time required for healing was signi cantly shortened in the ASC-microcarrier 6 complex group (t = 5.95, t t 0.01/2,4 , p 0.01); the time required for healing was also signi cantly shortened in the simple ASC treatment group as compared with the thread-drawing treatment group (t = 11.88, t t 0.01/2,4 , p 0.01). There were no signi cant differences in the body weight between the different groups. During the observation period, all anal stulas in the ASC-microcarrier 6 complex group, simple ASC treatment group and thread-drawing procedure group healed. When compared with the thread-drawing procedure group, the time required for healing was signi cantly shortened in the ASC-microcarrier 6 complex group (t = 5.95, t t 0.01/2,4 , p 0.01); the time required for healing was also signi cantly shortened in the simple ASC treatment group as compared with the thread-drawing treatment group (t = 11.88, t t 0.01/2,4 , p 0.01).
The ASC-microcarrier 6 complex group exhibited a longer time to heal, when compared to the simple ASC treatment group, but the difference was statistically insigni cant (t = 1.81, t t 0.05/2,6 , p 0.05).

Pathological examination
After three weeks of anal stula treatment, the original anal stula tissue was removed, under general anesthesia, for pathological section. In the control group, the anal stula was not healed, abundant necrotic tissue was observed in the stula, and considerable lymphocyte in ltration was observed with HE staining (Fig. 2d). All anal stulas in the experimental groups healed, with the presence of brous granulation tissues growing at the original anal stula site. HE staining was performed on the resected original anal stula site. Low lymphocyte accumulation was observed around the microcarrier 6 in the ASC-microcarrier 6 complex group (Fig. 2a). This may be attributed to the fact that the ASC-microcarrier 6 complex was not yet completely absorbed by the surrounding tissues, given the short observation period, and thus immune-stimulation was produced at the local tissues. Pathological sections from the simple ASC treatment group were found to contain many lipid vesicles (Fig. 2b), which may be due to the replacement of the stula tract by adipose tissues differentiated from ASCs. Pathological sections from the thread-drawing procedure group showed granulation tissues and lymphocyte in ltration (Fig. 2c), which might be caused by reactive hyperplasia of tissues surrounding the stula tract under stimulation from the thread.
4. ASC-microcarrier 6 transplantation for anal stula mainly promotes tissue regeneration through multi-directional differentiation, and it has been found that PI3K/AKT signaling pathway molecules are signi cantly enriched In this study, a whole-genome expression pro ling chip was tested, and high-throughput RNA sequencing was performed to screen for differentially expressed genes. After data analysis, differentially expressed genes were observed in the ASC transplantation group and ASC-microcarrier 6 composite treatment group, compared with normal control tissues. These genes were mainly concentrated during differentiation of various tissue types, such as striated muscle, vascular smooth muscle, and epithelial cells. The differential genes are mainly concentrated in the construction and differentiation of striated muscle. Tissue regeneration for stula repair was promoted via multi-directional differentiation. Further functional enrichment analysis of differential genes (KEGG pathway) indicated that PI3K/AKT signaling pathway molecules were signi cantly enriched, suggesting that the PI3K/AKP signaling pathway may play an important role in anal stula treatment.

Gene expression pro le difference GOterm analysis
shows that ASC -microcarrier 6 promotes muscle/striated muscle differentiation and contraction Gene expression pro le differences between the anal stula control group and ASC-microcarrier 6 treatment group were analyzed using the GOterm relationship network. The results suggested that ASCmicrocarrier 6 transplantation can promote differentiation, development, and contraction of muscles in anal stula ( Fig. 3 AB).

Discussion
Anal stula is an infectious disease occurring in the perianal region. The conventional thread-drawing procedure can ensure a satisfactory healing rate, despite the drawbacks regarding large postoperative wound surfaces and increased healing time. If the sphincter ani is severely damaged during surgery, patients may suffer from fecal incontinence, which greatly impairs quality of life [2].
ASCs are a type of mesenchymal stem cell, which are abundant in the human body, can be easily harvested, and have multi-directional differentiation, self-replication ability, and strong immune tolerance, as well as the ability to suppress CD4 + T cells [15]. Ideal applied stem cells can be the best source for stem cell transplantation and treatment [16]. ASCs can secrete a variety of growth factors, cytokines, antiin ammatory factors, and chemokines [17]. These anti-in ammatory factors and chemokines may play an important role in defending the immune response against infectious in ammation.
Therefore, ASCs can be used as seed cells to treat anal stula, promote wound tissue repair, and wound surface healing. It is becoming a research hotspot in injury repair and regenerative medicine. Analysis of all clinical research data employing foreign stem cell treatments for anal stula indicate that allogeneic adipose stem cells are advantageous due to being minimally invasive, reducing anal sphincter injury, reducing pain, and decreasing hospitalization [18]. Jiang Bin et al. reported the use of autologous adipose stem cells to treat 23 patients with complex anal stula. The clinical study has veri ed the effectiveness and safety of autologous stem cell transplantation for the treatment of anal stula. The total cure rate was 69.57%, of which 11 cases were Crohn disease anal stula with a cure rate of 90.91%, 12 cases of glandular anal stula exhibited a cure rate of 50% [19]. In terms of experimental research, Ferrer et al. established an animal model of Crohn's disease anal stula in dogs, and then treated them with human mesenchymal stem cells. The e cacy and safety were observed. No rejection occurred. All dogs' stulas healed within three months. Fistula recurred in two dogs, with a cure rate of 66.67% [20].
Several scholars have also suggested introducing a novel stula treatment in animal models. Volpe BB et al. attached mesenchymal stem cells to absorbable sutures and achieved signi cant results in the treatment of rabbit intestinal stula models, which displayed a signi cant improvement over sutures alone [21]. To date, only a small sample of retrospective studies have been conducted involving the use of ASCs in the treatment of anal stula. Research for anal stula treatment methods is still at the exploratory stage, and no consensus has been reached concerning the origin of adipose tissues of ASCs, separation and culture work ow, or injection dose and frequency.
It has been found that the use of scaffolds can improve the survival rate and biological function of transplanted cells. A scaffold provides a reticular structure, to which the stromal cells and ASCs adhere to form a microstructure. Injection of ASCs into lesions lead to micro-environmental changes of the anal stula, thereby producing a synergistic effect on the treatment.
Experiments show that autologous ASC transplantation can improve the prognosis of anal stula. A comparison of pathological sections from the ASC-microcarrier 6 complex treatment and the simple ASC treatment indicated that a small number of lymphocytes in ltrated the tissues near the microcarrier 6, possibly due to the incomplete absorption of microcarrier 6 by the surrounding tissues. Thus, the amount of microcarrier 6 can be reduced depending on the number of transplanted ASCs, while local physiotherapy can be applied to the transplanted site to lower the reactivity of local tissues to the transplant.
We also performed whole-genome expression pro ling microarray testing and high-throughput RNA sequencing to screen differentially expressed genes. The data indicated that the ASC-microcarrier 6 combined treatment group expressed differential genes compared to the normal controls. These genes were primarily concentrated during differentiation of various types of tissues, such as striated muscle, vascular smooth muscle, epithelial cells, and nerve cells. The differential genes are mainly concentrated in the construction and differentiation of striated muscle. Tissue regeneration for stula repair was promoted via multi-directional differentiation. Further, KEGG pathway data indicated that PI3K/AKT signaling pathway molecules were signi cantly enriched, suggesting that the PI3K/AKP signaling pathway may play an important role during anal stula healing. S Lin et al. found that insulin-like growth factor 1 (IGF-1) can promote endothelial cells/fat by activating the PI3K/AKT signaling pathway by observing vascular formation in stem cell co-culture systems [22]. H Zhou reports that the PI3K/AKT signaling pathway promotes paracrine functions of ASCs [23].

Conclusion
Our experiments show that the transplantation of ASCs together with microcarrier 6 is a therapeutic approach with high potential for the treatment of anal stula: microcarrier 6 can promote ASCs differentiation and the rapid healing of anal stula in vivo. However, we further need to unveil the complete mechanism of action of our approach. Furthermore, we also plan to improve the effect of ASCs transplantation for the treatment of anal stula via optimization of microcarrier 6 concentration and degradation rate, and via modi cation of PI3K / Akt signaling. Last but not least, the full characterization of ASCs + microcarrier 6 safety and long-term e cacy, very important for future translation to the clinical context, will be our next step.  b) Pathological sections from the simple ASC treatment group were found to contain many lipid vesicles (black arrow). c) Pathological sections from the thread-drawing procedure group showed tissue granulation and lymphocyte in ltration (black arrow). The red arrow indicates collagen. d) HE staining revealed in ltration of many lymphocytes in the control group. The left area exhibits brokeratoma necrosis, the middle area depicts lymphocyte in ltration, and the right area depicts bers and collagen.