In 1985, Erich Mühe[14] initially described a novel surgical technique, laparoscopic cholecystectomy, as a less invasive procedure to cure symptomatic cholelithiasis and cholecystitis. Through decades of practice and research, laparoscopic cholecystectomy has become the preferred option for treating symptomatic cholelithiasis and cholecystitis[15,16]. However, accompanying the development of laparoscopic cholecystectomy, the incidence of BDI of the laparoscopic technique seemed to be higher than that of the open technique[17]. Current studies claimed that the laparoscopic technique for biliary disease was associated with an increased number of BDIs[1,4]. The end-to-end anastomosis of the BD is regarded as the optimal approach for treating BDI. Nevertheless, there remains a high incidence of reconstructive failure due to unrepairable BD tissue defects. Thus, as an alternative option, Roux-en-Y choledochojejunostomy has been widely used to address the above conditions by effectively reconstructing the bilioenteric anatomy. However, the option has only improved the repair of the pathology of BDIs, which still shows a certain incidence of complications caused by the reflux of intestinal contents into the biliary tree, including cholangitis, strictures, bile leaks and a relatively high risk of cholangiocarcinoma[6]. Repairing the BDI while preserving the function of the Oddis sphincter remains a challenge for hepatobiliary surgeons.

In recent years, the development of novel materials has brought a new era for extrahepatic BDI. An extensive review of the literature shows that several materials have been used as scaffolds to treat BDIs. In 2002, Gómez et al[18] found that thin-walled FEP-ringed Gore-Tex vascular grafts were useful in the repair of BD injuries, which was the first report of an artificial BD in the field of BD repair. After that, several synthetic materials were applied to the manufacture of artificial BDs. However, the further application of the artificial BD in clinical trials was limited by the mechanical strength of the original materials as well as the molding technique. A report from Li et al[19] demonstrated a novel exploration of biosynthetic material by the experimental repair of BD defects in pigs using collagen scaffolds loaded with human collagen-binding bFGF. The short-term experimental results showed good immunocompatibility. Nevertheless, factors including a complicated manufacturing process and difficulty in making tubes may limit the application of this material to clinical trials.

In addition, several studies have performed animal research in which defects of the BD were patched with autogenous tissue in an attempt to avoid immunological rejection. The autogenous tissues included autogenous veins, single-layer small intestinal submucosa, etc. Although definite superiority in regard to compliance and immunocompatibility was observed, their further clinical utility was restricted by the mismatch of the diameter and thickness as well as the complicated procedure of the acquisition and preparation of autogenous materials[11,20]. From a pathophysiological point of view, animal-derived materials are similar in compliance and structure compared with those of humans. Thus, theoretically, animal-derived materials could present improved immunocompatibility following decellularization by removing cellular antigens. The similarity in diameter, thickness, flexibility and strength leads to advantages in the ease of anastomosis and the matching of different tissues as well as in the preservation of the Oddis sphincter function in preventing reflux cholangitis. A homologous animal-derived decellularized ureteral graft was explored in a BD defect porcine model[12]. However, only 3 mo of observation was carried out accompanied by unsatisfying results of the patency of the graft without T-tube or a silicone stent inserted. In addition, the strict requirement of homology also limits further exploration in clinical applications. Thus, the long-term observation of the efficacy and safety of heterogenous animal-derived materials in repairing BD remains unclear.

In the present study, we investigated the long-term replacement effect of ada-BD for two types of extrahepatic BD segmental injury models in the pig model. We chose ada-BD because it showed a high similarity between the bovine ureter and BD in the following ways: (1) A similar pathological structure (mucosa, muscular layer and outer membrane); (2) A similar sterile environment (with only slight differences in pH value and composition); (3) A similar physiological pressure (ureteral internal pressure 7-13 cmH₂O, physiological pressure of the CBD 12 cmH₂O); and (4) Similar in diameter, thickness and compliance. The result of immunological rejection is the primary cause of the induction of graft loss after transplantation[21,22]. Thus, the immunogenicity of grafts is an urgent problem that must be resolved. In this work, the immunogenicity of the bovine ureter was effectively removed via the technique of Triton-X-100/phosphate buffered saline decellularization. Subsequently, strengthening crosslinking associated with anticalcification was carried out with a 0.20-2.0 wt% glutaraldehyde solution to improve the mechanical properties, thermal stability and anticalcification feature. Finally, an animal-derived BD scaffold was manufactured for use in this study.

Overall, the duration of the operation and the BD anastomosis were basically the same as in human surgery. Cheng et al[12] established an animal BD damage and repair model by resecting a 1 cm long CBD followed by bridging the defect using a 2 cm long decellularized ureteral graft without tension. However, this animal experiment only considered the tension of anastomosis without considering the increased risk of biliary stricture caused by the fold of the redundant BD after the operation.

Different from previous studies, the present damage and repair model was developed with a 2 cm-to-2 cm replacement using the ada-BD to avoid longitudinal anastomotic tension and redundant BD tissue. The initial four animals in group A underwent ada-BD-to-duodenum anastomosis to repair the distal defect of the CBD and were sacrificed at the predetermined date of 6 mo postoperatively. Among these animals, the main complications, including bile leakage (1/8) and incision infection (1/8), were cured by conservative treatment without weight loss. The resulting increase in ALT on PoD 7 in group A was associated with liver function damage caused by bile leakage. Macroscopic assessments indicated that the ada-BD appeared to induce the covering of the visceral layer of the peritoneum and the regeneration of the biliary mucous membrane without the formation of any scars or strictures. As supplementary evidence, histological analysis (hematoxylin/eosin staining and cytokeratin 19 immunostaining) showed that the inner wall of the graft was covered by regenerated BD epithelium without marked inflammatory cell infiltration.

The above observations suggest that no obvious post transplantation rejection or biliary infection occurred in the experimental animals. With regard to the extrahepatic BD, the smooth muscle in the submucosal layer is a vital component that maintains the structural integrity, ensures the rigid structure and participates in the process of bile secretion. The desmin-positive area detected revealed the significant regeneration of smooth muscle in group A. Simultaneously, the remarkable regeneration of connective tissue in the submucosal layer was confirmed by Masson’s trichrome staining.

In comparison, the remaining four long-term observation animals recovered uneventfully after the operation. In addition, the histological analysis suggested the superiority in group B compared to group A regarding the regeneration of the BD epithelium and connective tissue. Notably, improved regeneration of the BD smooth muscle was observed in group A compared with group B. This phenomenon indicated that ada-BD-to-duodenum anastomosis showed a better induction effect on the regeneration of the BD smooth muscle compared with ada-BD-to-CBD anastomosis. No effect on the patency of the extrahepatic BD was found according to the macroscopic assessment and cholangiography. Thus, a prolonged investigation is needed to observe the long-term patency of the ada-BD graft. The enhancement of the regeneration of connective tissue in the submucosal layer in group B was confirmed by Masson’s trichrome staining. Both the macroscopic and microscopic assessment of the neo-extrahepatic BD showed no significant differences from a normal duct at 12 mo. Apparently, based on the results above, the ada-BD, as a nonimmunogenic scaffold, could provide good physiological conditions for the regeneration of autologous BD structures. This would suggest that the ada-BD was successfully used for the repair of BD segmental defects and achieved a similar effect as autologous BD end-to-end anastomosis.