Application Of DAT-ALG Hydrogel Loaded With Dental Pulp Mesenchymal Stem Cells In The Treatment Of Perianal Fistula In Crohn’s Disease

Objective:Crohn’s disease（CD）is an inflammatory bowel disease characterized by intestinal inflammation and fistula,and its incidence rate is increasing year by year.Crohn’s perianal fistula is a common serious complication of CD,characterized by long-term or intermittent recurrence,which seriously affects the prognosis and quality of life of patients and has the risk of changing the anal sphincter and creating a permanent stoma.At the moment,the treatment is primarily a combination of bacteriostasis and anti-inflammatory drugs,immunosuppression,and surgery,but the cure rate remains low,patients’quality of life has not improved,and it also places a significant burden on society.The purpose of this study was to explore the effect of hydrogel loaded with dental pulp mesenchymal stem cells（DPMSCs）on the course and prognosis of Crohn’s perianal fistula and further explore the relevant mechanisms,so as to provide a new treatment for Crohn’s perianal fistula.Methords:1)The stemness of DPMSCs was detected by flow cytometry and induced differentiation in vitro.2)Ultra-pure decellularized adipose tissue（DAT）was prepared from adipose tissue through acellular and enzymatic digestion.Oil red O staining,Masson staining,DAPI staining,scanning electron microscopy,and DNA content detection were used to look at DAT’s microstructure,how well it removed cells,and how much collagen it kept.3)DAT and alginate（ALG）were crosslinked in the presence of an appropriate Ca²⁺concentration to form a DAT-ALG injectable interpenetrating network hydrogel.The related properties and biocompatibility of the hydrogel were analyzed by gel experiments,scanning electron microscopy,and live/dead cell staining.4)The SD rats were used to construct the anal fistula model,and the DAT-ALG hydrogel loaded with DPMSCs was injected for treatment.The weight change,perianal secretion,stool bleeding,stool viscosity,inflammatory factor expression level,tissue HE staining,immunohistochemical staining,and other comprehensive evaluations of the treatment effect were detected.5)Transmission electron microscopy（TEM）,nanoparticle tracking analysis（NTA）,and western blot（WB）were used to find out how hydrogel affected stem cell exosome secretion.This was done to learn more about the possible molecular mechanism of DAT-ALG hydrogel loaded with DPMSCs in the treatment of Crohn’s perianal fistula.Results:1)Flow cytometry showed that DPMSCs highly expressed stem cell-related markers,and the results of induced differentiation in vitro showed that DPMSCs had the ability to undergo multi-directional differentiation.2)After acellular treatment,fat tissue can effectively remove lipids and cells and retain most of its collagen,indicating that DAT has been successfully obtained.3)The results of the gel experiment showed that DAT-ALG hydrogel could form gel successfully at body temperature.The scanning electron microscope showed that DAT-ALG hydrogel had a good three-dimensional structure,and the results of living/dead cell staining showed that DPMSCs in the hydrogel could survive successfully,indicating that the hydrogel had no obvious cytotoxicity to stem cells.4)The results of the rat perianal fistula model showed that the combined transplantation of DAT-ALG hydrogel and DPMSCs could effectively improve the body weight of SD rats,improve the expression level of inflammatory factors in the peripheral blood and tissues around the fistula,inhibit the secretion of the perianal fistula,and improve local inflammation,thereby promoting fistula healing.5)TEM and NTA tests showed that the size and shape of the exosomes secreted by DPMSCs were not significantly changed by hydrogel,WB test showed that the expression of exosome markers in DPMSCs loaded with DAT-ALG hydrogel was higher.Conclusion:This study confirmed that DPMSCs have stem cell-related characteristics.DAT-ALG hydrogel has a good three-dimensional structure,which can support the survival and growth of DPMSCs,and there is no obvious cytotoxicity.DPMSCs loaded onto DAT-ALG hydrogel can effectively improve the expression of Crohn’s perianal fistula-related inflammation and peripheral blood inflammation-related factors,thereby promoting the healing of Crohn’s perianal fistula.This therapeutic effect may be related to DAT-ALG promoting the secretion of DPMSCs-exosomes.This study provides a novel treatment method for Crohn’s perianal fistula based on stem cells.