Preliminary Study On Tissue Engineering Materials Of Anal Fistula Suppository Based On Silk Fibroin-chitosan

Purpose: 1. To prepare a new type of polymer material silk fibroin-chitosan(SFCS). To study the optimal dosage proportion in line with tissue engineering requirements of the two materials through representation observation and degradation test. 2. To perform biological compatibility experiment by using SFCS composite material and to conduct preliminary evaluation of its feasibility as a medical polymer material in the application of anal fistula suppository.Method: 1. Prepare SFCS composite stents; adopt calcium chloride-alcohol-water system(Ca CL2 : C2H5OH:H2O=1:2:8) for dissolution. 2% SF solution will be obtained after dialysis, concentration and centrifugation. Prepare 3% CS solution by dissolving chitosan into acetic acid. Mix the two in different proportions to obtain finished product of SFCS 3D stent after die-filling, freeze drying and crosslinking.2. Representation experiment of materials: Adopt scanning electron microscope test method to observe characteristics of the stent material, and calculate the swelling ratio, water absorption and porosity.3. Static degradation test: Add SFCS composite material into phosphate buffered solution(PBS solution). Put the solution into a constant-temperature oven with the temperature of 37℃. Observe the degradation rate and stability of SFCS.4. Perform evaluation of cell biocompatibility of SFCS composite material. Perform hemolytic test for SFCS. Implant SFCS stent and rubber spline into thigh muscle of SD rats. Put the rats to death at day 7, day 14, day 21 and day 28 and perform HE staining and Masson three-color staining respectively.Results: 1. SFCS stents with different components will be obtained after mixing of silk fibroin and chitosan solution at different mass ratio.2. The porosity of SFCS with different proportions is higher than 95%. The swelling ratio, water absorption of the materials will increase along with increasing of chitosan proportion.3. The in vitro degradation experiment indicates that with the increasing of silk fibroin, the degradation rate of composite material will decrease successively.4. Animal studies show that compared with SFCS stents, the interior inflammatory cells of patients within the control group almost have disappeared and have been filled with normal cells. Complete fibrous capsule wall structures have been formed. The collagen content is obviously higher than that of the control group. The collagenous fiber is more thick and tight, with more obvious tendency.Conclusion: 1. Mix the silk fibroin and chitosan. Prepare 3D porous stent with stable structure successfully after freeze drying. The 40%SF-60%CS group will obtain the most appropriate porosity, satisfactory swelling ratio and water absorption. Thus it can be choose as the preferred proportion for biological stent researching.2. In animal implanting experiments, SFCS composite material has excellent biocompatibility with appropriate in vivo degradation rate. It is a good alternative reference for early stage of materials research of anal fistula suppository, and is expected to become an ideal material for research of anal fistula suppository.