Biological Characteristics Studies Of A New Biodegradable Meterial For Stent And Animal Study Of Intraluminal Stent Repair Of Bile

PART I Synthesis of Poly [sebacic acid-co-(1,3-propanediol)-co-1,2-propanediol)] elastomers and manufacture of anastomosis stentsObjective To synthesize a new type of bio-degradable high molecular polymer, to develop and manufacture a series of anastomosis stents for hollow viscus.Method It was synthesized using1,3-propanediol,1,2-propanediol and sebacic acid with the molar ratio of2:3.75:1.25by Two Step Melt Polycondensation Synthetic Method, then purified by anhydrous methanol. The molecular weight of the material synthesized was determined by gel permeation chromatography(GPC). And the material was identified according to the Infrared Spectroscopy (IR) spectra. The mould was modified on the basis of original design model, and the stents were manufactured by injection molding with the novel material.Result We successfully synthesized the target product Poly [sebacic acid-co-(1,3-propanediol)-co-1,2-propanediol)](PSPP) by Melt Polycondensation Synthetic Method, with Weight-average Molecular Weight of about67000, Number-average Molecular Weight of about30000and Distribution Width of2.2. There were four absorption peaks including1161cm-1,1728cm-1,2927cm-1and2851cm-1in the Infrared Spectroscopy spectra. Then we successfully manufactured a series of anastomosis stents with PSPP by injection molding for hollow viscus.Conclusion With sebacic acid,1,3-propanediol and1,2-propylene glycol as monomers, we successfully synthesized PSPP by Melt Polycondensation Synthetic Method, and manufactured a series of anastomosis stents by injection molding for hollow viscus. PART Ⅱ Experimental study of the biocompatibility of PSPPObjective To systematically assess the biocompatibility of the novel bio-degradable polyester PSPP in vitro and in vivo, providing experimental base for further animal experiments and clinical researches.Method Based on the medical equipment biology assessment guide promulgated in china (GB/T16886.2008), we chose cytotoxicity test, Ames test as items of in vitro experiments and systemic toxicity tests, intradermoreaction, delayed type hypersensitivity, implant test as well as rectal mucous membrane stimulation test as items of in vivo experiments.Result (1) In vitro experiments:Cytotoxicity test demonstrated that RGR results in24h,48h and72h are all above75%, the scale was1. Ames test demonstrated that the colonies numbers in bacterial strains TA97, TA98, TA100and TA102in PSPP group were similar to those of the control group with no statistical difference (P>0.05), and no more than2times that of the control group, the result was negative.(2) In vitro experiments:After intradermal injection of PSPP leach liquor, all experimental animals had a primary stimulate scoring of0in24h,48h and72h. Systemic toxicity tests:After tail vein injection of PSPP leach liquor in experimental group and saline in control group, observations in4h,24h,48h and72h all showed no death in both groups, and animals in both group ate normally, without symptoms such as vomiting, dull-looking, hypocinesis or dyspnea. Animals gained weight in both groups, and there was no significant difference. Delayed type hypersensitivity:After injected by PSPP leach liquor and PSPP leach liquor combining with Freund's complete adjuvant, assessing skin allergy score on the third day and seventh day after allergic sensitization,24h and48h after stimulation showed0,0,0,0respectively. Rectal mucous membrane stimulation test:After one week continuous rectal infusion of PSPP leach liquor, both macroscopic observation and histopathological examination showed no manifestation of congestion, swelling or necrosis on the rectum mucosa. Implant test:Two weeks after implantation, gross specimen adjacent to the stent revealed mild surrounding tissue edema in both experimental group and control group. Results in four weeks and twelve weeks were similar in both groups, with stent surrounded by a thin layer of bursa wall, no obvious adhesion to surrounding tissues, no manifestation of congestion, swelling or necrosis. In histological, the inflammatory cells reaction level was Ⅳ and capsule chamber reaction level was IV in both groups two weeks after implantation the inflammatory cells reaction level was Ⅲ and capsule chamber reaction level was Ⅱ in both groups four weeks after implantation; the inflammatory cells reaction level was Ⅰ and capsule chamber reaction level was Ⅰ in both groups twelve weeks weeks after implantation.Conclusion The above results demonstrate that PSPP has satisfactory biocompatibility both in vitro and in vivo. PART Ⅲ Biodegradability of PSPP in vitro and in vivoObjective This study is to investigate the biodegradation of PSPP in vitro and vivo, the main factors that contribute to the degradation rate of PSPP, the degradation time of PSPP in different part of gastrointestinal.Method In vitro degradation we chose human fresh bile, simulated gastric and intestinal liquid as media, the PSPP stent as sample. Each individual media has48samples. Each stent was soaked in flat-bottomed test tube containing10ml degradation media which was changed daily, with37℃,100rev/min sustained oscillation. We took three samples from each media every week to observe their appearance and integrity, to measure remaining weight and weight-average molecular weight, and to study the morphology of the surface by scanning electron microscopy. The observation period is four months or to the disintegration of sample. The in vivo degradation use Bama miniature pig as experimental subject, every five samples as a group are placed in the stomach, small intestine and colon. We killed pigs and removed the sample on a regular basis (group every four days in small intestine group and a week in other groups) to observe morphological changes, weight average molecular weight changes, and study the morphology of the surface by scanning electron microscopy.Result In vitro fresh bile, the degradation cycle was about3.5months, the total weight loss was nearly60%, weight-average molecular weight was reduced to about31,000from the original67000, scanning electron microscopy revealed the sample surface gradually showed a fish scale-like change with shedding. In simulated gastric liquid, the appearance of sample kept intact, weight, weight-average molecular weight and the morphology of the surface nearly unchanged. In simulated intestinal liquid, the degradation cycle was about2.5months, the total weight loss was nearly46%, weight-average molecular weight was reduced to about30,000, scanning electron microscopy revealed the sample surface gradually showed a fish scale-like change. In small intestine group, the degradation cycle was about2weeks, weight-average molecular weight unchanged, scanning electron microscopy revealed the sample surface was obviously corroded. In gastric group, the thickness of sample did not change significantly in6weeks, weight average molecular weight decreased slightly, scanning electron microscopy revealed the sample surface became rough with a small amount of hole. The samples in the colon degradated in about5weeks, Weight average molecular weight decreased gradually, scanning electron microscopy revealed the sample surface was corroded.Conclusion In vitro in human fresh bile, the degradation cycle was about3.5months, In simulated intestinal liquid, the degradation cycle was about2.5months, In simulated gastric liquid, the degradation cycle was over4months. The alkaline environment contributed to the degradation of PSPP, and in the acidic environment, PSPP performance was relatively biologically inert. The emulsifying function of bile and trypsin played an import role to improve the degradation of PSPP. In vivo, The degradation rate of PSPP was fast in the small intestine, the degradation time is about2weeks, which match the healing time of the anastomotic stoma in small intestine. In the colon, the degradation time is about5weeks; In the gastric, PSPP's degradation was very slow, performed relatively inert.PART Ⅳ Animal study of intraluminal stent repair of bile duct injury with omentum Objective To assess the feasibility and safety of intraluminal stent repair with omentum for bile duct thermal burn together with perforation.Methods A total of32Bama minipigs were randomly chosen and divided into two groups,16in each. Experimental models with common bile duct (CBD) injury (thermal burn together with perforation) were built by animal surgery, the experimental group was repaired by a intraluminal stent with greater omentum, and the control group was repaired by suture. The incidence of jaundice and bile leakage was evaluated in both groups. Animals were sacrificed at2weeks,1,3and6months after operation. The healing of anastomosis was observed. The status of scar formation, cholangiography, histologic findings by HE, Masson staining and electron microscope were evaluated. The expression of a-SMA, TGF-β1and b-FGF were also evaluated and compared.Result All operation were accomplished successfully. No obvious leakage happened in either experimental or control group. The change of postoperative liver function bilirubin value were comparison analysised, it found that two weeks and1month after operation the ALP change in the control group was significantly greater than the experimental group,3months after operation, the AST change was significantly higher in the control group.6months postoperation, the DBiL change was significantly higher in the control group. Cholangiography indicates that there was no restenosis in experimental group, in control group, there was one animal with linear narrow in the anastomosis with choledochectasia3months after operation, and2animals with cholangiectasis in the proximal duct and intrahepatic bile duct with scar stricture in the anastomosis6months after operation. Both HE stain and Masson stain showed that inflammatory reaction and hyperplasia of the bile duct wall in the experimental group was much more moderate than that in control group. Immunohistochemistry stain showed that the experimental group had less expression of a-SMA and TGF-β1while more expression of b-FGF than that of control group. Fluorescence quantitative PCR results show that2weeks after operation, the experimental group had slightly increased expression of TGF-β1and b-FGF mRNA compared to the control group, and less express of a-SMA mRNA; After one month, b-FGF mRNA had increased expression while TGFβ-1and a-SMA mRNA had less expression compared to the control group;3months after operation, b-FGF, a-SMA and TGF-β1mRNA express in the experimental group had less expression, especially TGF-P1mRNA;6months after operation, the expression of b-FGF and a-SMA mRNA were similar in the two groups, while TGF-β1mRNA expression was less in the experimental group.Conclusion Intraluminal stent covered with greater omentum is a safe and feasible approach of repair of bile duct injury, it could reduce binary restenosis of bile duct.