| Objective:Anal fistula is a tubular pathologic passage perforating the rectum,anus and skin,which mainly caused by perianal abscess.Frequent infection can result in the formation of irregular branching structures of fistula,which often require radical surgical treatment.However,the surgery will inevitably damage the anal sphincter,resulting in serious postoperative complications such as fecal incontinence.Since there is no damage to perianal tissue,fistula filling is one of the promising minimally invasive treatments.Currently,the common filling materials in clinic contain fibrin glue and anal fistula plug.Due to the rapid gelation of fibrin glue,during the process of injection,the rapidly formed fibrin clot is easy to block the fistula,resulting in the difficult of continue injection.And the anal fistula plug is not injectable and is not suitable for filling the muti-branch irregular fistula.In addition,the implantation of filling materials can easily provide an invasion route for resident bacterium in the skin and the digestive tract,causing infection and even sepsis.Therefore,it is of great clinical value to explore a new fistula filling material with injectable and antibacterial activity.Cryogel is a gel matrix formed by cross-linking polymer precursors in freezing environment and melting at room temperature.Due to its spongy macroporous structure and good mechanical elasticity,cryogel can be applied as a 3D scaffold for filling the confined and localized areas.However,cryogel needs to be prefabricated,which limits its application in anal fistula treatment.Recently,a large number of literatures have reported that microgel prepared by mechanical fragmentation of bulk gel has the unique physical properties such as shear thinning and self-healing,and can undergo liquid-solid dynamic transformation,which overcomes the defects of poor shape adaptability of cryogels.Thus,microgel has great potential for fully filling the irregular structure fistula by injection.Therefore,we propose the assumption:preparing cryogel by cross-linking polyethylene glycol diacrylate(PEGDA),which has good biocompatibility and economy,with N,N’-bis(acryloyl)cysteamine(BAC),containing disulfide bonds,in the below zero environment to form the interconnected macroporous structures.Then,microgel is generated by mechanical fragmentation to develop a novel filling material for sealing irregular structure fistula.Meantime,because it’s loaded with silver nanoparticles,microgel owns the antibacterial activity,so as to control wound infection and promote tissue healing.Based on this,this study will focus on the morphology,rheological and mechanical properties,biodegradability,antibacterial activity and histocompatibility of the filling material,and finally evaluate its therapeutic effect in animal anal fistula models.Methods:1.Observing the microstructure of cryogels and microgels.The microstructure of cryogels and microgels and the coating of dopamine and silver nanoparticles were observed by scanning electron microscope(SEM).The elemental composition of the filling material was analyzed by energy dispersive spectrometer(EDS).2.Testing the compressive elasticity and rheological properties of cryogel and microgels.The difference of the compressive mechanical strength of cryogel,hydrogel and derived microgel was compared through the single compression tests.The resilience and tolerance of cryogel were tested by cyclic compression tests.Shear thinning and selfhealing properties of microgel were verified by rheological tests.3.Detecting the in vitro and in vivo degradation properties,antibacterial activity and histocompatibility of microgels.The changes of the quality and morphology of the filling material were observed by soaking in the reductive degradation solution and subcutaneous injection in rats.The antibacterial activity of the microgel loaded with the silver nanoparticles(Ag NPs)was detected by k-B drug sensitivity test.Moreover,the biosafety of the filling material was further evaluated by the cytotoxicity test and in vivo test.4.Evaluating the therapeutic effect of microgels in the animal anal fistula models.Quantitatively evaluating the performance of infection control and tissue proliferation repair through H&E staining and inflammatory cell grading.By immunohistochemical staining,CD31 and α-SMA were employed to label the neovascular epithelial cells and fibroblasts,respectively,to evaluate the acceleration of tissue repair and wound healing by filling materials.Results:1.Cryogel has been confirmed to have micrometer-scale interconnected macroporous structure.High concentration of crosslinker can increase the crosslinking density and reduce the pore size.In addition,microgel prepared by mechanical fragmentation still retain similar macroporous structure to cryogel.Energy dispersive spectrometer confirmed the presence of silver element in microgel-DA-Ag NPs group.2.Cryogel has good compression elasticity and cannot be damaged at compression of50% at least.Compared with hydrogel,cryogel has higher compressive mechanical strength,which can be further enhanced by increasing the concentration of crosslinker.During the amplitude scanning,the microgel underwent a sol-gel transition and showed a typical shear-thinning behavior.In shear rate scanning,the viscosity of microgel decreased with the increase of shear rate.These characteristics were beneficial for injecting of microgel.In addition,dynamic strain change step tests showed that microgel exhibited self-healing properties by cyclic shearing at low strain(1%)and high strain(20%),which can prevent the microgels from extrusion under tissue compression.3.The new filling material showed a tendency of degradation in vivo and in vitro,and the degradation rate of the material could be controlled by adjusting the concentration of crosslinker.The microgel loaded with silver nanoparticles(0.5 mg/m L)showed obvious antibacterial activity against Escherichia coli and Staphylococcus epidermidis,and showed no obvious cytotoxicity and rejection reaction in vitro and in vivo,demonstrating good histocompatibility.4.In the treatment of animal anal fistula model,H&E staining and inflammatory cell grading results demonstrated that,compared with the control group,microgel-DA-Ag NPs group could effectively control infection.In CD31 and α-SMA immunohistochemical staining,the neovascularization decreased significantly and the fibroblasts increased significantly in microgel-DA-Ag NPs group,which illustrated that the wound repairing was obviously accelerated and the proliferation of fibroblasts had changed from early angiogenesis to later fibroblast.Conclusion:PEGDA-based cryogel microgel has good mechanical properties,which can fully fill the fistula with irregular multi-branched structure by injection.Meanwhile,microgel loaded with nano-silver particles(0.5 mg/m L)has good biocompatibility and broadspectrum antibacterial activity,which can effectively control infection and promote wound healing. |
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