Satoshi Kaihara

Silicon micromachining to tissue engineer branched vascular channels for liver fabrication

To date, many approaches to engineering new tissue have emerged and they have all relied on vascularization from the host to provide permanent engraftment and mass transfer of oxygen and nutrients. Although this approach has been useful in many tissues, it has not been as successful in thick, complex tissues, particularly those comprising the large vital organs such as the liver, kidney, and heart. In this study, we report preliminary results using micromachining technologies on silicon and Pyrex surfaces to generate complete vascular systems that may be integrated with engineered tissue before implantation. Using standard photolithography techniques, trench patterns reminiscent of branched architecture of vascular and capillary networks were etched onto silicon and Pyrex surfaces to serve as templates. Hepatocytes and endothelial cells were cultured and subsequently lifted as single-cell monolayers from these two-dimensional molds. Both cell types were viable and proliferative on these surfaces. In addition, hepatocytes maintained albumin production. The lifted monolayers were then folded into compact three-dimensional tissues. Thus, with the use microfabrication technology in tissue engineering, it now seems feasible to consider lifting endothelial cells as branched vascular networks from two-dimensional templates that may ultimately be combined with layers of parenchymal tissue, such as hepatocytes, to form three-dimensional conformations of living vascularized tissue for implantation.

Anatomical variations and surgical strategies in right lobe living donor liver transplantation: lessons from 120 cases.

Background. Anatomical variations in right liver lobe are common. However, clinical implications and surgical management of these variations in living donor liver transplantation have not been analyzed systematically. Methods. Surgical anatomy of vascular and biliary structures in 120 right lobe grafts were reevaluated by reviewing the results of preoperative (computerized tomography and Doppler ultrasonography) and intraoperative (cholangiography) imaging as well as surgical findings. The data were analyzed in relation to surgical management of anatomical variations. Results. The incidence of variants leading to multiple portal vein anastomoses was 7.5%. The incidence of dual right hepatic veins was 0.8%; 30% of the grafts had significant accessory hepatic veins (>5 mm) and 13.9% of these were multiple. All of them were successfully reconstructed with technical modifications including venoplasty and venous grafts, except for two cases with multiple intraparenchymal portal vein branches to the anterior segment. The incidence of dual hepatic arteries was 1.7%, but only one of them was reconstructed without negative sequelae. The incidence of variants potentially leading to multiple bile duct anastomoses was 35.0%, and eventually 39.2% of the grafts had multiple orifices. With a variety of techniques including ductoplasty, hepaticohepaticostomy, and biliary stent, total incidence of leakage and stenosis was 10.8% and 9.2%, respectively. Although ductoplasty, internal stent or no stenting, seemed to be associated with increased risk of complications, anatomical variants, multiple bile ducts, and duct-to-duct reconstruction did not bear a significant risk. Conclusions. Anatomical variations of vascular and biliary structures in right lobe grafts are common. However, most can be managed safely with technical modifications. Only cases with intraparenchymal origin of the anterior portal vein(s) may form a relative contraindication, especially when combined with similar biliary variants. Otherwise, intraoperative assessment of biliary anatomy was enough for successful management. Detailed and precise assessment of vascular and biliary anatomy is vital for appropriate surgical management.

Living-donor liver transplantation for hepatocellular carcinoma.

In cadaveric liver transplantation, the Milan criteria have been accepted as the selection criteria for hepatocellular carcinoma (HCC) patients in considering organ allocation. However, the situation is different in living-donor liver transplantation (LDLT), in which the donor has a strong preference for altruism. The authors describe herein their experience with LDLT for HCC patients using their patient selection criteria. From February 1999 to March 2002, right lobe LDLT was performed in 56 patients with HCC. The authors’ exclusion criteria included only those with extrahepatic metastasis or vascular invasion detected during preoperative evaluation. Thirty patients (54%) were in tumor, node, metastases stage IVa and 25 patients (45%) did not meet the Milan criteria at the time of LDLT. The follow-up period was 1 to 39 months (median, 11 months). The overall survival rates at 1 and 3 years were 73% and 55%, respectively, and the latter was significantly lower than that of adult right lobe LDLT without HCC (71% at 3 years). Fourteen patients died because of postoperative complications without tumor recurrence. Thirty-six patients survived without recurrence and six patients had recurrence. Among the six patients with recurrence, four had survived for 11 to 36 months after LDLT. In the analysis of patients who survived longer than 3 months after transplantation, 19 of 20 within the Milan criteria survived without recurrence. However, 15 of 20 patients beyond the criteria also survived without recurrence for 3 to 33 months (median, 12 months) and three of five patients with recurrence were alive for 11 to 36 months (median, 20 months). Histopathologic grading and microscopic portal venous invasion had significant negative impact on tumor recurrence. LDLT was an effective treatment for uncontrollable hepatocellular carcinoma. Because many patients who did not meet the Milan criteria survived without tumor recurrence after transplantation, different patient selection criteria are necessary in LDLT to save those with advanced HCC.

Duct-to-Duct Biliary Reconstruction in Living Donor Liver Transplantation Utilizing Right Lobe Graft

ObjectiveTo assess the feasibility and safety of duct-to-duct biliary anastomosis for living donor liver transplantation (LDLT) utilizing the right lobe. Summary Background DataBiliary tract complications remain one of the most serious problems after liver transplantation. Roux-en-Y hepaticojejunostomy has been a standard procedure for biliary reconstruction in LDLT with a partial hepatic graft. However, end-to-end choledochocholedochostomy is the technique of choice for biliary reconstruction and yields a more physiologic bilioenteric continuity than can be achieved with Roux-en-Y hepaticojejunostomy. The authors performed right lobe LDLT with end-to-end duct-to-duct biliary anastomosis, and this study assessed retrospectively the relation between the manner of reconstruction and complications. MethodsBetween July 1999 and December 2000, 51 patients (11–67 years of age) underwent 52 right lobe LDLTs with duct-to-duct biliary reconstruction and remained alive more than 1 month after their transplantation. Interrupted biliary anastomosis was performed for 24 transplants and the continuous procedure was used for 28. A biliary tube was inserted downward into the common bile ducts through the recipient’s cystic duct in 16 transplants (cystic drainage), or a biliary stent tube was pushed upward into the anastomosis through the cystic duct in four transplants (cystic stent), or upward into the anastomosis through the wall of the common bile duct in 31 transplants (external stent). ResultsBiliary anastomotic procedures consisted of 34 single end-to-end anastomoses, 11 double end-to-end anastomoses, and 7 single anastomoses for double hepatic ducts. Overall, 5 patients developed leakage (9.6%) and 12 patients suffered stricture (23.0%). For biliary anastomosis with interrupted suture, the incidence of stricture was significantly higher in the cystic drainage group (53.3%, 8/15) than in the stent group consisting of cystic stent and external stent (0%, 0/8). While the respective incidences of leakage and stricture were 20% and 53.3% for intermittent suture with a cystic drainage tube (n = 15), they were 7.7% and 15.4% for a continuous suture with an external stent (n = 26). There was a significant difference in the incidence of stricture. ConclusionsDuct-to-duct reconstruction with continuous suture combined with an external stent represents a useful technique for LDLT utilizing the right lobe, but biliary complications remain significant.

Impact of recipient age on outcome of ABO-incompatible living-donor liver transplantation.

Background. Transplantation of hepatic grafts from ABO-incompatible donors is controversial because of the risk of hyperacute rejection mediated by preformed anti-ABO antibodies. The aim of the present study was to evaluate the outcome of liver transplants performed with ABO-incompatible living-donor livers and to detect risk factors for development of complications. Methods. From June 1990 to February 2000, 66 patients, 10 months to 55 years old (median, 2 years old), received 68 ABO-incompatible living-donor liver grafts. The antibody titer and clinical course were followed prospectively during a period ranging from 3 to 11 years. Results. The 5-year patient survival was 59%, 76%, and 80% for ABO-incompatible, ABO-compatible, and ABO-identical grafts, respectively (P <0.01). In patients <1 year old, ≥1 to <8, ≥8 to <16, and and ≥16 years old, 5-year survival was 76%, 68%, 53%, and 22%, respectively. The incidence of intrahepatic biliary complications and hepatic necrosis in ABO-incompatible living-related grafts (18% and 8%, respectively) was significantly (P <0.0001) greater than in ABO-compatible and ABO-identical grafts (both 0.6% and 0%, respectively). Predictive risk factors for increased mortality and morbidity were age greater than 1 year and elevated anti-ABO titers before transplantation. Conclusions. ABO-incompatible liver transplantation was carried out with relative safety in infants <1 year old but was not satisfactory in children >1 year in long-term follow-up. Patients aged >8 years remain at considerable risk of early fatal outcome because of hepatic necrosis, and new strategies to prevent antibody-mediated rejection are required.

Dynamic seeding and in vitro culture of hepatocytes in a flow perfusion system.

Our laboratory has investigated hepatocyte transplantation using biodegradable polymer matrices as an alternative treatment to end-stage liver disease. One of the major limitations has been the insufficient survival of an adequate mass of transplanted cells. This study investigates a novel method of dynamic seeding and culture of hepatocytes in a flow perfusion system. In experiment I, hepatocytes were flow-seeded onto PGA scaffolds and cultured in a flow perfusion system for 24 h. Overall metabolic activity and distribution of cells were assessed by their ability to reduce MTT. DNA quantification was used to determine the number of cells attached. Culture medium was analyzed for albumin content. In Experiment II, hepatocyte/polymer constructs were cultured in a perfusion system for 2 and 7 days. The constructs were examined by SEM and histology. Culture medium was analyzed for albumin. In experiment I, an average of 4.4 X 10(6) cells attached to the scaffolds by DNA quantification. Cells maintained a high metabolic activity and secreted albumin at a rate of 13 pg/cell/day. In experiment II, SEM demonstrated successful attachment of hepatocytes on the scaffolds after 2 and 7 days. Cells appeared healthy on histology and maintained a high rate of albumin secretion through day 7. Hepatocytes can be dynamically seeded onto biodegradable polymers and survive with a high rate of albumin synthesis in the flow perfusion culture system.

Surgery-related morbidity in living donors of right-lobe liver graft: lessons from the first 200 cases.

Background. Living‐donor liver transplantation (LDLT) using the left lateral segment or left‐lobe graft has been widely accepted, but currently, right‐lobe grafts are more commonly used in many LDLT programs with yet unknown risks for donors. Methods. We investigated our initial 200 donors of righ‐lobe grafts to focus on the incidence and variety of surgery‐related morbidity. Changes in liver function tests were also analyzed to clarify the relation with donor age, steatosis of the liver, and residual liver volume (RLV). Complications were surveyed for a median period of 28.7 months. Results. In all the donors, liver enzymes and bilirubin were normalized within 1 month. Enzymes on day 1 were significantly higher in donors with older age, macrovesicular steatosis, and larger RLV. Bilirubin on day 1 was significantly higher in donors with smaller RLV. Biliary enzyme was not normalized in the majority at 1 month after donation. Seventy‐five complications occurred in 69 donors. Biliary complications were most common, which consisted of 26 bile leakages (13%) and 3 biliary strictures (1.5%) in 27 donors. No significant dependence of the incidence was observed either for donor age (≥50 years), body mass index (BMI) (≥25 kg/m2), estimated RLV (<40%), or medical history. None of the complications led either to mortality or to long‐term sequelae. Conclusions. Complications occurred in a significant proportion of right‐lobe donors irrespective of donor age, BMI, estimated RLV, and medical history. Living‐liver donor surgery requires more care in right‐lobe transplants.

Living-donor liver transplantation with monosegments.

Background. Living-donor liver transplantation is now an established technique to treat children with end-stage liver disease. Implantation of left-lateral segment grafts can be a problem in small infants because of a large-for-size graft. We report 10 cases of transplantation using monosegment grafts from living donors. Method. Of 506 children transplanted between June 1990 and June 2002, 10 patients (median age 196 days, median weight 5.9 kg) received monosegment living-donor liver transplants. The indication for using this technique was infants with an estimated graft-to-recipient weight ratio of over 4.0%. Results. Graft and patient survival was 80.0%. There were no differences in donor operation time and blood loss between monosegmentectomy and left-lateral segmentectomy (n=281). Monosegmental transplantation had a high incidence of vascular complications (20.0%). Conclusion. Monosegmental living- donor liver transplantation is a feasible option with satisfactory graft survival in small babies with liver failure.

Short- and long-term donor outcomes after kidney donation: analysis of 601 cases over a 35-year period at Japanese single center.

Background. The lack of deceased donors in Japan means that living-donor kidney transplantation is necessary in as many as 80% of cases. However, there are few data on perioperative complications and long-term outcome for live kidney donors. Methods. To determine associated perioperative morbidity and long-term mortality among live kidney donors, we reviewed 601 donor nephrectomies performed at our institution between 1970 and 2006 and attempted to contact all of the donors (or their families) to ascertain their present physical status. The survival rate and causes of death were compared with an age- and gender-matched cohort from the general population. Results. Although three donors (0.5%) experienced major perioperative complications, that is, femoral nerve compression, pulmonary thrombosis, and acute renal failure, all of the donors recovered and left hospital without complications. Among 481 donors (80%) for whom details were available at the time of inspection, 426 (88.5%) were still surviving. Donor survival rates at 5, 10, 20, and 30 years were 98.3%, 94.7%, 86.4%, and 66.2%, respectively. The mean interval between kidney donation and death was 183±102 (7–375) months, and the mean age at death was 70±11 years. The survival rate of kidney donors was better than the age- and gender-matched cohort from the general population, and the patterns and causes of death were similar. Conclusions. Our data suggest that continuation of living-donor kidney transplantation programs is justified in short- and long-term donor safety.

Long-term follow-up of tissue-engineered intestine after anastomosis to native small bowel

BACKGROUND Our laboratory has investigated the fabrication of a tissue-engineered intestine using biodegradable polymer scaffolds. Previously we reported that isolated intestinal epithelial organoid units on biodegradable polymer scaffolds formed cysts and the neointestine was successfully anastomosed to the native small bowel. The purpose of this study was to observe the development of tissue-engineered intestine after anastomosis and to demonstrate the effect of the anastomosis over a 9-month period. METHODS Microporous biodegradable polymer tubes were created from polyglycolic acid. Intestinal epithelial organoid units were harvested from neonatal Lewis rats and seeded onto the polymers, which were implanted into the abdominal cavity of adult male Lewis rats followed by 75% small bowel resection (n=24). Three weeks after implantation, the unit/polymer constructs were anastomosed to the native jejunum in a side-to-side fashion. The anastomosed tissue-engineered intestine was measured by laparotomy 10, 24, and 36 weeks after the implantation (n= 14). During the laparotomy, all rats with an obstruction in their anastomosis were killed and excluded from the statistical analysis. Another five rats were also killed at 10 and 36 weeks for histological and morphometric studies. RESULTS All analyzed rats survived this study and significantly increased their body weight by 36 weeks. Obstruction of the anastomosis was observed in one rat at 24 weeks and in two rats at 36 weeks; however, the anastomosis was patent in the other 11 rats by 36 weeks. The tissue-engineered intestine of these 11 rats increased in length and diameter at 10, 24, and 36 weeks after anastomosis; there were statistically significant differences between each time point except between the length of 10 and 24 weeks (P<0.016 by Wilcoxon signed rank test). Histologically the inner surface of the tissue-engineered intestine was lined with well-developed neomucosa at 10 and 36 weeks; however, there were small bare areas lacking neomucosa in the tissue-engineered intestine at 36 weeks. Morphometric analysis demonstrated no significant differences in villus number, villus height, and surface length of the neomucosa at 10 and 36 weeks. CONCLUSIONS Anastomosis between tissue-engineered intestine and native small bowel resulted in no complications after operation and maintained a high patency rate for up to 36 weeks. The tissue-engineered intestine increased in size and was lined with well-developed neomucosa for the duration of the study.