Preparation And Preliminary Application Of Anastomotic Tube Based On Tissue Engineering By Intra-colon In The Form Of Intra-colonic Bypass

In order to reduce the incidence and severity of consistent dead complications after anastomotic leakage in colorectal surgery,this thesis attempts to use tissue engineering degradable medical polymer materials,and to prepare and screen the matching of performance matching with 3 different manufacturing processes.The tube,in conjunction with the method of achieving intra-colonic bypass,assists in the healing of the anastomosis after colorectal surgery.The purpose of the anastomosis tube prepared in this study is to help reduce the complications of anastomotic leakage as much as possible,save more lives,improve the quality of life after surgery,and unnecessary stoma and subsequent secondary retraction surgery,which is expected to improve.The level of prevention and treatment of anastomotic leakage in digestive surgery.This dissertation consists of the following 3 parts:Part I Preparation,evaluation and screening of intestinal anastomosis tubes based on tissue engineering polymer in the form of intra-colonic bypassObjective:In order to reduce the consistent complication of anastomotic leakage after gastrointestinal resection,a tissue engineering polymer substrate suitable for assisting intra-colonic bypass in the form of biodegradability,tissue compliance,and biocompatibility is selected;Three different methods of 3D printing and melt injection were used to prepare the anastomosis tube,evaluate the characteristics of various anastomotic tubes and screen appropriate protocols to assist the healing of anastomotic stoma after digestive tract and prevent anastomotic leakage.Methods:Two polymers were prepared in this study:polylactic acid（PLA）,polylactic acid glycolic acid（PLGA）;two amphiphilic diblock copolymers:（polyethylene glycol monomethyl ether-block-poly-L-Lactide block copolymer（m PEG-b-PLA）and polyethylene glycol monomethyl ether-block-poly-L-lactide-co-glycolide block copolymer（m PEG-b-PLGA））.Using these different properties of the material as the substrate,according to the established specifications of the local environment of the intestine to be reconstructed and anastomosed,three different techniques of electrospinning,3D printing and melt injection were used to prepare the anastomosis tube for assisting anastomotic healing.At the same time,the physical properties of the materials such as hydrophilicity,microtubule morphology,chemical composition,degradation behavior and other key indicators were characterized and optimized.Results:Results 1:In this study,16 kinds of PLA and PLGA with different molecular weight and different group distribution ratios were synthesized by optimized preparation process.Different molecular weights and different groups were assigned to 6 kinds of m PEG-b-PLA and m PEG-b-PLGA.Perform physical and chemotherapy characterization;Results 2:Using the above polymer materials as raw materials,different types of characteristics of the anastomosis tube were prepared by electrospinning,3D printing and melt injection molding;the anastomosis tube prepared by PLGA was poor in hydrophilicity.（Water contact angle is 107°±0.8°）,after the introduction of the PEG segment,the m PEG-b-PLGA substrate（where m PEG-b-PLGA₃ has a contact angle value of 65°±1.0°）The hydrophilic properties of the anastomosis tube have been improved.Secondly,the anastomosis tube prepared by the electrospinning process has a microscopic structure formed by superposing layers of nanofibers and having a microporous structure（the gap size is from 2000 to 6000 nm.The diameter of the nanofiber filaments constituting the film is 660 to 760 nm）It has good hydrophilicity,degradability and tissue compliance.Conclusions:The anastomosis tube prepared by 3D printing and melt injection using PLGA and PLA is hard and has poor flexibility,which is not conducive to suture fixation.It is prepared by electrospinning using synthetic m PEG-PLGA amphiphilic diblock copolymer as substrate.The anastomosis tube has more suitable physical and chemical properties,biodegradability,biocompatibility and tissue compliance with respect to 3D printing and melt injection molding,and is suitable for the development of the second animal experiment.The anastomosis tube prepared by 3D printing and melt injection using PLGA and PLA is hard,which is not conducive to operation fixation such as surgical suture,and the tissue compliance cannot meet the synchronous peristalsis of the intestine;using synthetic m PEG-b-PLA_1/2/3,m PEG-PLGA_1/2/3 amphiphilic diblock copolymer as substrate,the anastomosis tube prepared by electrospinning process has more suitable physical and chemical properties,biodegradability and biological phase than 3D printing and melt injection molding process.Capacitive,organizational compliance.After summing up various factors,m PEG-b-PLGA₃ was selected as the material,and the anastomosis tube was made by electrospinning,which was proposed for the second animal experiment.PartⅡ:Preliminary application of intestinal anastomosis tube based on tissue engineering in the form of intra-colonic bypassObjective:The biocompatibility of the intestinal anastomosis tube prepared by the electrospinning process using m PEG-b-PLGA₃ was investigated.SD mice with cecal disconnection-anastomosis were used as the research object to test whether the material and anastomosis operation were safe and convenient;whether it would help to reduce the short-term mortality,the incidence of anastomotic leakage,and the medium-and long-term related concurrency Symptoms,can promote the healing of anastomotic;whether the anastomosis tube has advantages in the reconstruction of the digestive tract.Methods:（1）Cell biology evaluationThe cell biocompatibility evaluation of the intestinal anastomosis tube prepared by the electrospinning process of m PEG-b-PLGA₃ was carried out.Focus on the blood compatibility and cytotoxicity of the tube.The blood compatibility of the tube was evaluated by direct contact with the blood in the anastomosis tube material.The epithelial cell culture and the anastomosis tube were used to observe the adhesion and cell growth under the microscope,and stained with fluorescein diacetate（FDA）.Growth and survival status.（2）Experimental mice evaluation105 male SD mice of common grade,and the cecal disconnection-anastomosis model was constructed without mechanical preparation and without oral and intravenous antibiotics.The first stage evaluated whether the anastomosis tube was effective and safe in the short term:two groups of SD rats（n=60）were treated with cecal disconnection-anastomosis.In the control group（n=30）,after the cecum was dissected,the absorbable line was sutured at the anastomosis with a 6-needle intermittent suture;the anastomosis group（n=30）,after the cecum was disconnected,the m PEG-b-PLGA₃ was used as the material for electrospinning.The anastomosis tube with the inner diameter of the same cecum was placed in the intestinal lumen at the anastomosis,and the anastomosis was performed by anastomosis.After the operation,the rats in the two groups were given free access to food and water immediately after waking.Compare the time required for the two groups of anastomosis,mortality.Survival rats in each group were subjected to exploratory laparotomy on the 7th day after operation:abdominal adhesion index and anastomotic blast burst pressure test were performed to observe the HE pathological staining of the anastomosis and evaluate the inflammation.The second stage evaluated whether the anastomosis tube caused intestinal obstruction and anastomotic stenosis in the long-term in vivo:normal SD rats（n=45）,and the first stage of anastomosis anastomosis operation and perioperative management,respectively Laparotomy was performed 2 weeks after surgery（n=15）,4 weeks（n=15）,and 6 weeks（n=15）.Observe the diameter of the anastomosis and observe the diameter of the intestine at 5 cm above the anastomosis.Results:Results 1.The hemolysis rates of the anastomotic tubes prepared by m PEG-b-PLA₂,m PEG-b-PLA₃,m PEG-b-PLGA₂ and m PEG-b-PLGA₃ as main substrates were:0.5%,1.1%,0.8%,respectively.And 1.5%.Compared with the commercialized PLGA（3%）material without introducing m PEG,the anastomosis tube prepared by the material prepared by the subject has a lower hemolysis rate.The results of cell co-culture experiments showed that the synthesized polymer substrate had low cytotoxicity,and the cultured cells were able to proliferate and migrate on the surface of the substrate.After culture,at the bottom of the cell culture plate,the epithelial cells have covered the entire cell plate.Due to the large distribution of hydrophilic PEG segments on the surface,the biocompatibility is improved,which is more conducive to epithelial cells.Adhesion,growth and proliferation.Results 2.The first stage of animal experiment:the time of surgical anastomosis was shown in the two groups.The control group was 16.95±1.83 minutes,which was longer than the 13.89±2.02 minutes of the anastomotic tube group.There was a statistical difference（P<0.001）,suggesting an anastomosis.With the auxiliary support of the tube,the operation time can be shortened to some extent.Six mice（20.0%）died in the control group.The cause of death was anastomotic leakage,abdominal infection,and peritonitis.One patient had intra-abdominal hemorrhage.One died in the anastomotic group（3.3%）.The non-anastomotic leakage died.The cause of suturing operation blocked the intestinal tract and caused death of mechanical intestinal obstruction.There was a significant difference in death between the two groups（P<0.001）.The laparotomy was performed 7days after surgery,and the abdominal adhesion of the control group was 1.67±0.844significantly higher than that of the anastomotic tube group（1.23±0.728）（P<0.001）.The limited intra-abdominal adhesion caused by early leakage of the anastomosis was considered.Subsequently,the cecum was taken and the burst pressure test of the abdomen was performed in the abdomen.The control group was 145.88±13.339 mm HG significantly lower than the anastomosis group 172.97±11.947 mm HG（P<0.001）.HE staining of the anastomotic site of the control group showed that the inflammatory cell score was 3.67±0.93,and the anastomotic group was 2.90±0.82（P<0.001）.The second stage of animal experiment:the dilatation or stenosis of the intestine near the anastomosis was observed at 2,4,and 6 weeks.With the prolongation of the time after the application of anastomotic anastomosis,the anastomotic diameter measurements were:2.27±0.234cm,2.22±0.214cm,2.33±0.241cm,and there was no obvious abnormality between the diameters of the anastomosis（P=0.410）;there was no significant dilatation and stenosis at 5 cm above the anastomosis:2.37±0.154 cm at 2 weeks,2.29±0.181cm at 4 weeks,2.32±0.221 cm at 6 weeks,（P=0.507）.Conclusions:.（1）The hemolysis rate of the prepared polymer material in this subject is less than 3%,and m PEG-b-PLGA₃ is 1.5%.The cultured cells are able to proliferate and migrate on the surface of the anastomosis tube,the cells survive well,and the coagulation shows satisfactory results.（2）Two-stage animal implantation results show that the biodegradable anastomosis tube is safe and effective in the process of healing the cecal anastomosis in rats.The operation time of the anastomosis operation was significantly reduced.It was considered that the suture process was facilitated by the anastomosis tube support,which facilitated the convenience and feasibility.The implantation of the anastomotic tube can effectively prevent the occurrence of anastomotic leakage and significantly reduce the perioperative mortality;Abdominal adhesions,considering the isolation of the anastomotic tube to protect the localized peritonitis caused by a small A-level anastomotic leakage that is difficult to avoid by the 6-needle suture method;also enhanced the anastomotic burst pressure;significantly reduced Infiltration of local inflammatory cells.Moreover,with the prolongation of postoperative time,there was no dilatation and stenosis of the intestine at the anastomosis and above the anastomosis,which had no effect on the survival and eating of the animals,and achieved time-dependent degradation.Considering the intra-colonic bypass for the isolation of the anastomosis tube,providing a relatively clean,low-tissue local micro-environment for the healing of the intestinal end,which contributes to the good healing of the anastomosis and reduces the related mortality and complications;The anastomosis tube has a strong advantage in the reconstruction of the digestive tract.The third part:"the status quo and research progress of anastomotic reconstruction and healing",the review.