Study On Exosome-loaded Hydrogel Adhesive For Promoting Intestinal Anastomosis Healing

Background:As a serious complication of gastrointestinal surgery,postoperative anastomotic leakage can lead to increased mortality,delayed postoperative recovery,prolonged hospitalization,and potential delays in adjuvant therapy for cancer patients.It is estimated that at least one million patients worldwide experience anastomotic leakage-related complications after gastrointestinal surgery every year.Therefore,improving the quality of anastomotic healing is quite essential for the prevention anastomotic leakage,and surgeons can use a range of methods,including improved surgical techniques,preoperative mechanical bowel preparation,and oral antibiotics to prevent the leakage postoperatively.Unfortunately,more than half of patients still do not benefit from these interventions.Consequently,promoting intestinal anastomotic healing and minimizing the occurrence of anastomotic leakage has become a challenging area of research in gastrointestinal surgery.In recent years,researchers have discovered that mesenchymal stem cell-derived exosomes(^MSCExos)can mimic the function of MSCs in tissue regeneration while avoiding the safety risks and ethical constraints associated with stem cell transplantation,which had great promise for clinical application in regenerative medicine.However,the role of ^MSCExos in the field of anastomotic repair remains unknown.Therefore,this study focused on the effects of administration of exogenous ^MSCExos on intestinal anastomosis healing.The traditional mode of ^MSCExos administration（intravenous or local injection）has various disadvantages,such as poor bioavailability and low tissue retention,which hugely limits the application of ^MSCExos in gastrointestinal anastomosis repair.An ideal strategy is to encapsulate ^MSCExos in a biomaterial carrier,through which ^MSCExos is slow-released locally into the intestinal anastomosis,aiming to maximize the therapeutic effect.In recent years,medical biomaterials represented by hydrogels have been widely used in regenerative medicine,and many encouraging results have been achieved in the effective loading and long-lasting slow release of ^MSCExos.Consequently,to explore new methods to improve the quality of anastomotic healing,a hydrogel adhesive loaded with ^MSCExos was constructed in this study and applied to a rat intestinal anastomosis model.It was hypothesized that the successfully prepared hydrogel adhesive can effectively adhere to the intestinal anastomosis locally and promote anastomotic healing by slow release of ^MSCExos.To this end,human umbilical cord MSC-derived exosomes(^uMSCExos)was extracted,a novel hydrogel adhesive that can be used to localize intestinal anastomoses in this study was constructed,and its effects on the anastomosis healing through ex vivo experiments and ^uMSCExos-miRNA microarrays were explored.Most importantly,the effects and possible mechanism of ^uMSCExos-miRNA microarrays on anastomotic healing were explored,and a new theoretical basis and research direction for the future application of ^MSCExos and biomaterials-based combination therapy in anastomotic repair was provided as well.Methods:1.Extraction and characterization of human umbilical cord mesenchymal stem cell（uMSC）and ^uMSCExos:Firstly,primary uMSC was isolated from umbilical cord using tissue block apposition method and then identified by apposition morphology,flow cytometry,and triple lineage-induced differentiation（osteoblastogenic,lipogenic,and chondrogenic）experiments.Afterwards,the culture supernatant of uMSC was collected and ^uMSCExos that was characterized by transmission electron microscopy,nanoparticle tracking analyzer,and protein blotting for morphology,particle size,and signature proteins was extracted using differential ultracentrifugation.2.Preparation and characterization of collagen（Col）/tannic acid（TA）hydrogel adhesive-^uMSCExos/Col/TA adhesive loaded with uMSCExos:Col/TA adhesive that prepared by physical cross-linking was characterized by scanning electron microscopy,infrared spectroscopy,rheometer,tilt assay,and visceral adhesion assay to characterize its physicochemical properties.BCA method was used to detect the slow-release properties of ^uMSCExos and ^uMSCExos uptake assay by co-culturing with L929 cells.Besides,in vitro biocompatibility and regeneration potential of ^uMSCExos/Col/TA adhesive was achieved by live/dead cell staining and CCK-8.The safety of the ^uMSCExos/Col/TA adhesive in vivo was assessed by degradation performance test,blood biochemical test and visceral HE staining.3.The effects of ^uMSCExos/Col/TA adhesive on the healing of rat intestinal anastomosis:A rat intestinal anastomosis model was established and randomly divided into 4groups,namely PBSgroup,Col/TA group,^uMSCExos group,and ^uMSCExos/Col/TA group,to treat the anastomosis.The rats were executed on the 7th day after the operation,and the changes of body weight and feces and the general conditions,such as anastomotic leakage and abdominal adhesion,were observed in each group.The anastomotic intestines were taken for the detection of anastomotic burst pressure and hydroxyproline content,HE staining,Masson staining,ELISA,immunohistochemistry and immunofluorescence staining,etc.,after which the effects of the ^uMSCExos/Col/TA adhesive on the healing of the intestinal anastomosis in the rats were evaluated.4.^uMSCExos-miRNA microarray assay and bioinformatics analysis:miRNA microarray assay and bioconfidence analysis of ^uMSCExos were performed to preliminarily explore the miRNAs that may play a role in intestinal anastomosis healing by ^uMSCExos and their mechanisms.Results:1.The results of uMSC adherent morphology,flow identification of stem cell markers and three-lineage induced differentiation experiments are as follows:（1）uMSC showed a typical MSC morphology under the microscope;（2）uMSC highly expressed MSC positive markers CD105,CD73 and CD90,and lowly expressed negative markers CD34,CD45 and HLA-DR;（3）uMSC could form osteoblasts,chondrocytes and adipocytes after differentiation and induction of culturing in vitro.Additionally,the results of transmission electron microscopy,nanoparticle tracking analyzer and protein blotting are as follows:（1）^uMSCExos had the typical spherical and cup-shaped morphology of exosomes;（2）the particle size（138.4 nm）and the expression of signature proteins（TSG101,CD9）of ^uMSCExos met the criteria of exosomes.Totally,the above experimental results proved that ^uMSCExos was successfully extracted.2.The prepared ^uMSCExos/Col/TA adhesive was in the form of light-yellow gel.The scanning electron microscopy results revealed that the Col/TA adhesive had a three-dimensional pore-like structure,and the surface of the ^uMSCExos/Col/TA adhesive contained a large number of exosomes.The rheological results illustrated that the Col/TA adhesive possessed good viscoelastic properties,and the infrared spectral map of the Col/TA adhesive had the characteristic infrared spectral peaks after cross-linking of Col and TA hydrogen bonds.The results of tilt and visceral adhesion experiments demonstrated that Col/TA adhesive had strong adhesion properties.According to the exosome release experiments,^uMSCExos/Col/TA adhesive could release ^uMSCExos slowly for more than 7 days.The results of co-cultivation experiments with L929 cells showed that ^uMSCExos/Col/TA adhesive had good adhesive properties.Col/TA adhesive could be efficiently taken up by the cells.Based on CCK-8 proliferation assay,^uMSCExos/Col/TA adhesive could increase the proliferation viability of uMSC,fibroblasts L929,vascular endothelial cells HUVEC,intestinal epithelial cells Caco-2,and macrophages Raw264.7 in vitro.The above experimental results indicated that hydrogen-bonded cross-linked ^uMSCExos/Col/TA adhesive had been successfully prepared,which can effectively slow the release of ^uMSCExos and had good bioactivity and pro-regenerative potential in vitro.The results of degradation performance test,blood biochemical test and HE staining of internal organs suggested that the ^uMSCExos/Col/TA adhesive had a good safety profile in the absence of significant toxic side effects.3.^uMSCExos/Col/TA adhesive decreased the expression of metallo-matrix protease 9and increased the bursting pressure,hydroxyproline content,neoplastic granulation tissue thickness and collagen content of the intestinal anastomosis after it was applied to intestinal anastomoses in a rat model.Moreover,^uMSCExos/Col/TA adhesive increased the expression levels of multiple growth factors at the site of the anastomosis and promoted cell proliferation and angiogenesis.However,^uMSCExos/Col/TA adhesive decreased the expression of multiple inflammatory factors and reduced the inflammatory response and apoptosis in the anastomosis.On the other hand,^uMSCExos/Col/TA adhesive reduced the inflammatory M1-type macrophages in the anastomosis and increased the anti-inflammatory and pro-repairing M2 macrophages.The above experimental results indicated that ^uMSCExos/Col/TA adhesive could promote the healing of intestinal anastomosis in vivo.4.Based on ^uMSCExos-miRNA microarray assay,^uMSCExos was enriched with a variety of miRNAs that can promote intestinal repair,and 12 miRNAs that may play a role in intestinal regeneration were identified through screening.Conclusion:The pro-regenerative potential of ^uMSCExos and the wet tissue adhesion ability of Col/TA hydrogels were utilized to successfully construct a Col/TA adhesive loaded with ^uMSCExos,which demonstrated its efficacy and safety in promoting intestinal anastomotic healing.Our study proved that this combination therapy based on exosomes and biomaterials is a promising cell-free local delivery strategy,which could provide new research ideas and directions for the improvement of anastomotic healing.