Yasuko Toshimitsu

A Tissue‐Engineered Artificial Bile Duct Grown to Resemble The Native Bile Duct

The aim of this study was to fabricate an artificial bile duct for the development of a new treatment for biliary diseases. Eighteen hybrid pigs were implanted with a bile duct organoid unit (BDOU) made of a bioabsorbable polymer. Twelve of the transplanted BDOUs had been seeded with autologous bone marrow cells (BMCs) in advance. Six animals, the controls, were grafted with the scaffold alone with no BMCs seeded. The common bile duct was cut, the hepatic cut end of the native common bile duct was anastomosed to the BDOU and the other end was anastomosed to the duodenum. The controls underwent a similar operation. The neo‐bile duct was removed at pre‐determined time points and investigated histologically. All 18 recipient pigs survived until their sacrifice at 6 weeks, 10 weeks or 6 months. Histological examination revealed incomplete epithelialization of the neo‐bile duct at 6 weeks and 10 weeks after transplantation. At 6 months, the organoid exhibited a morphology almost identical to that of the native common bile duct. No differences were found between the controls and BMC‐seeded pigs. These results show that the artificial bile duct thus fabricated can serve as a substitute for the native bile duct.

A novel treatment for bile duct injury with a tissue-engineered bioabsorbable polymer patch

BACKGROUND With the recent widespread use of laparoscopic cholecystectomy and living-donor liver transplantation, complications involving the biliary system, and stenosis in particular, are encountered frequently. Although various invasive and noninvasive techniques are now available for the treatment of biliary stenosis, recurrence and other problems limit their value and utility. Our group sought to investigate whether a bioabsorbable polymer (BAP) patch could serve as a novel treatment for biliary stenosis. METHODS In anesthetized hybrid pigs (n = 12), a spindle-shaped portion of the lower common bile duct wall was excised, and a BAP patch was implanted at the excision site. The animals underwent repeat laparotomy at 5 weeks (n = 6) and at 4 months (n = 6) after implantation to recover the graft sites for gross and histologic studies. Blood chemistry was analyzed from samples taken during the patch implantation and recovery. RESULTS All of the recipient pigs survived until killing. All gained weight and showed no evidence of jaundice. The BAP-patched duct remained patent without obstruction at 5 weeks postimplantation. Blood chemistry did not reveal any increases in hepatobiliary enzyme activities. Histology showed accessory glandular structures in the neo-bile duct. At 4 months, the graft site was indistinguishable from the native duct. Intra-operative cholangiography revealed dilation of the patched site, but no dilatation of the intrahepatic bile ducts. Blood chemistry values were within normal ranges. Histology at the site of the patch confirmed the growth of a cuboidal columnar epithelium similar to that of the native duct. CONCLUSION The bile duct was dilated only focally at the site of implantation. This newly designed substitute has potential for application as a novel treatment for biliary injury and stenosis.

Hepatocyte dynamics in a three‐dimensional rotating bioreactor

Background and Aims:  The use of an artificial liver system with extracorporeal circulation or a three‐dimensional bioreactor perfused with liquid culture medium inevitably exposes hepatocytes to fluid mechanical stress (MS). The expression of liver‐specific hepatocyte functions seems to be modulated by the magnitude of MS. Nonetheless, few studies have focused on the direct effects of MS on hepatocytes. We subjected hepatocytes to MS using an MS loading device and investigated the effects on the cytoskeleton and hepatocyte dynamics inside three‐dimensional scaffolds by monitoring the changes in actin fiber, one of the components of the cytoskeleton. We also assessed the influence of MS on specific hepatocyte functions.

Tissue-Engineered Patch for the Reconstruction of Inferior Vena Cava During Living-Donor Liver Transplantation

In living-donor liver transplantation, only a portion of the donor’s liver is grafted into the recipient; therefore, if the hepatic vein and inferior vena cava (IVC) in the recipient fail to be transformed or dilated properly, it could cause inadequate blood flow from the liver graft to the IVC. We have developed an easy-to-use tissue engineered patch that can be used for the reconstruction of the hepatic vein and IVC. Five hybrid pigs (weighing 15–30 kg) served as the recipients of the patch. A bioabsorbable polymer sheet was used to produce the patch, with no cells seeded. The pigs were laparotomized, followed by the removal of a 3 × 2-cm portion of the infrahepatic IVC, which was then patched with the polymer sheet. Three months after the operation, the graft site was removed and subjected to gross and histologic examinations. All five pigs survived until they were killed 3 months after the operation. On gross examination, the polymer sheet grafted onto the IVC was completely absorbed, and the graft site was morphologically similar to the native IVC. In all five pigs, the patched IVC was free of stenosis or deformation. Immunohistochemical examination revealed that the patch site was lined with endothelial cells and that smooth muscle was present under the epithelium. Like the native IVC, the patch site tested positive for factor VIII. These findings suggest that this polymer sheet may be useful for the reconstruction of the IVC and hepatic vein during living-donor liver transplantation in humans.

Regeneration of extrahepatic bile ducts by tissue engineering with a bioabsorbable polymer

With the widespread adoption of laparoscopic cholecystectomy and living-donor liver transplantation in recent years, complications involving the biliary system, stenosis in particular, are increasing. Various invasive and non-invasive techniques are now available for the treatment of biliary stenosis, but all are compromised by a high risk of recurrence and other problems. As a potential solution, our group has developed a bioabsorbable polymer (BAP) tube for implantation as a bypass graft. In the study reported here, we implanted this BAP tube and confirmed bile duct regeneration at the graft site after the tube had been degraded and absorbed into the body. We briefly describe our findings on extrahepatic biliary tissue regeneration, focusing on the possibility of its clinical application. This artificial bile duct may promote the development of novel treatments for biliary disease.

Fatal impact of lymphocyte cross‐matching upon humoral rejection after adult living related liver transplantation

Although positive lymphocyte cross-match combinations of donor and recipient are rare, humoral rejection (HR) is still a serious problem after organ transplantation. The importance of lymphocyte cross-matching and human leukocyte antigen (HLA) histocompatibility have been reported for kidney and combined kidney-liver transplantation [1,2]. The role of anti-donor HLA antibodies in graft loss is also well-known [3]. However, it is generally believed that positive cross-match should not be considered a contraindication for liver transplantation (LT) [4]. We report a thought-provoking case of living donor liver transplantation (LDLT) with positive cross-match combination. A 46-year-old female suffered from well-developed cirrhosis caused by hepatitis C virus. Because of her deteriorating condition, she was referred to our division for LDLT. On admission, she was found to have a low-grade fever; also, cell counts in ascites and pleural effusion were increased. Spontaneous bacterial peritonitis and pleuritis were managed by drainage and cefotaxime. Infections were well-controlled preoperatively. Lymphocyte cross-match tests were performed using direct complement-dependent cytotoxicity and antihuman globulin assays [5,6]. Pretransplant results were positive. Recipient showed strong reactions against donor HLA Class I antigens, and the same immunoreactivity was confirmed by flow cytometry (FCM) (Fig. 1). We performed additional tests to assess antigen-specific immunoreactivity. Recipient’s lymphocytes showed strong immunoreactivity against Class I loci including B 55. HLA typing revealed that donor had this HLA B locus. ABO blood group was compatible. As we were unable to find a more suitable donor, we performed LDLT accompanied by splenectomy. Graft recipient weight ratio was 0.91. We used tacrolimus, methylprednisolone and mycophenolate mofetil as immunosuppressants. The postoperative course was uneventful until POD 3 when recipient experienced a sudden elevation of lactate dehydrogenase levels, a decrease in platelet count and severe fragmentation of red blood cells. Total bilirubin levels were increased after POD 3 leading to prolonged jaundice. On POD 4, X-ray showed acute respiratory distress syndrome-like condition. A diagnosis of HR was made, and other reasons were ruled out. Plasma exchange (PE) was performed daily after POD 4 and she received steroid pulse therapy from POD 5. Although immunoreactivity against Class I antigen was down-regulated during early postoperative period, it increased again from POD 6 (Fig. 1). On POD 8, peripheral blood examination showed evidence of hemolysis. Percutaneous micro-ecchymosis was noted and coagulation profiles were consistent with disseminated intravascular coagulation. Patient’s condition worsened and she did not respond to further treatment, including daily PE. Histopathologic examination by liver needle biopsy clearly showed severe graft damage. The patient died on POD 9 despite intensive treatment.

α-fetoprotein–producing Clear Cell Carcinoma of the Extrahepatic Bile Ducts

A 79-year-old woman visiting our hospital with chief complaints of epigastric pain and jaundice was emergently admitted. Her alpha-fetoprotein (AFP) level was as high as 2265 ng/mL at admission. Her abdominal computed tomography scan revealed dilation of the intrahepatic bile duct and a tumorlike lesion protruding into the cystic duct and gallbladder from the junction between the middle portion of the bile duct and the right and left hepatic ducts. Surgery revealed a tumor extending from the extrahepatic bile duct (EHBD) to the cystic duct, with no intrahepatic tumor components. The tumor was histologically diagnosed as an AFP-producing cholangiocarcinoma of the clear cell type, originating from the EHBD. None of the previously reported cholangiocarcinomas of the AFP-producing clear cell type have been confined to the EHBD or have been resectable in a curative manner without hepatectomy.

Liver resection using a soft-coagulation system without the Pringle maneuver.

BACKGROUND/AIMS The Pringle maneuver is generally performed to reduce the amount of blood loss during hepatic resection. We have developed a method to sufficiently control blood loss during hepatectomy without applying the Pringle maneuver. This study was performed to determine the safety and operative blood loss in hepatectomy performed by this new method. METHODOLOGY We performed 102 hepatic resections without the Pringle maneuver. We retrospectively compared the short-term operative outcome between these 102 cases and another 75 hepatic resections performed with the Pringle maneuver. The resections without the Pringle maneuver were performed using a soft-coagulation system. RESULTS The median length of the surgery using the soft-coagulation system without the Pringle maneuver was 135 minutes, significantly shorter than the surgical time required for resection with the Pringle maneuver 297 minutes (p<0.001). The median volume of operative blood loss was significantly lower in the non-Pringle-maneuver group (200cc vs. 704cc; p<0.001). Regarding postoperative liver function, AST, ALT, T-Bil and PT, levels were all significantly improved in the non-Pringle-maneuver group (p<0.01). CONCLUSIONS Our data suggest that hepatic resection using a soft-coagulation system without the Pringle maneuver is extremely safe and effective in controlling bleeding.

Colon Wall Regeneration Using a Bio-Absorbable Polymer Sheet

Postsurgical abdominal adhesions (PSA) are the predicted outcome for >90% of abdominal and GI track surgeries. Further, these adhesions account for up to 75% of small bowel obstructions. As such, healing of GI tract and peritoneal surfaces with subsequent adhesion formation remains a vexing clinical problem. Because PSAs are preceded by formation of a fibrin gel matrix (FGM), we hypothesized fibrin to be an ideal biomarker of pro-adhesive sites and that application of a fibrin-homing polymer during the initial stages of wound healing could interrupt the FGM and prevent PSA formation. Methods: Poly(ethylene glycolmethacrylate-block-methacrylic acid) (PEG-PMA) copolymers were synthesized and functionalized with fibrintargeting peptides sequence (CREKA) to generate a variety of molecular architectures. Gold-coated quartz crystal microgravimetry (QCM) was used to identify the polymers capacity to inhibit fibrin deposition. Their ability to inhibit cell adhesion was measured with an In Vitro cell binding assay. Subsequently, the polymers were tested in BALB/c mice using a “double injury” peritoneal/intestinal excision/window abrasion model. Results: CREKA-conjugated-polymers suppressed fibrin deposition in QCM assays and reduced cell adhesiveness compared to the sham controls in an In Vitro wound surface model. In addition, diblock PEG-PMA copolymers inhibited both fibrin deposition and cell attachment. The most effective polymers were then assayed In Vivo. Mice showed no significant clinical symptoms (e.g. appearance, weight) compared to controls, indicating no adverse effects of the copolymer administration. Furthermore, the polymer resulted in a significant reduction in the severity of adhesions (2.4±0.13 vs. 2.8±0.06, PEG-PMA-treated vs. controls, p=0.015). Conclusions: A fibrin-targeted blocking copolymer (PEG-PMA) reduced the severity of adhesions at the site of peritoneal/intestinal injury, suggesting the potential use of targeted polymers as post-surgical adhesion suppressing agents. Supporting grants NIHDE019496(TD) and NIH-DE19177(HO).

Extended Left Hepatic Lobectomy for Hepatic Hilar Bile Duct Cancer: A Novel Surgical Procedure in Which the Right Hepatic Duct Is Transected before the Hepatoduodenal Ligament Is Skeletonized

As a general principle, the procedure of isolating a cancer region from surrounding tissues (skeletonization) is not taken until the last stage of a cancer operation. This principle is often disregarded in surgery for cancers of the hepatic hilar duct, however, as it may be mandatory to skeletonize the region before resecting the liver. Our group invented ‘extended left hepatic lobectomy’, a new surgical procedure for hepatic hilar bile duct cancer, in which we isolate the liver and transect the right hepatic duct before skeletonizing the cancer region in the hepatoduodenal ligament.