Hirofumi Utsunomiya

Local delivery of basic fibroblast growth factor increases both angiogenesis and engraftment of hepatocytes in tissue-engineered polymer devices

Background. We investigated heterotopic hepatocyte transplantation on biodegradable polymers as a potential treatment for end-stage liver disease. The primary problem has been insufficient engraftment of transplanted cells partly because of insufficient vascularization. Increasing vascularization through locally delivered angiogenic factors may increase angiogenesis and hepatocyte engraftment. Methods. We studied the effect of local delivery of basic fibroblast growth factor (bFGF) on angiogenesis and hepatocyte engraftment within tissue-engineered liver constructs. Poly-l-lactic acid discs were fabricated and coated with either a mixture of saline, sucralfate, and Hydron (control group) or bFGF, sucralfate, and Hydron (bFGF group). bFGF release from polymers in vitro was tested using an ELISA. Hepatocytes were isolated from Lewis rats, seeded on control (n=9) or bFGF (n=11) polymers, and implanted into the small bowel mesentery of syngeneic animals. Specimens were harvested after 2 weeks and analyzed for hepatocyte engraftment. Microvascular density was compared between control (n=6) and bFGF groups (n=5). Results. Three hundred twenty-three thousandths of a microgram of bFGF were incorporated per polymer. Greater than 99% of the bFGF was released into solution by 72 hr in vitro. Two weeks after implantation, microvascular density, as measured by capillaries per high-powered field (c/hpf), was significantly greater in the bFGF group (43.8 c/hpf), compared with the control group (30.5 c/hpf;P <0.005). Specimens from the bFGF group (mean engraftment, 61,355 &mgr;m2) showed a 2.5-fold increase in hepatocyte engraftment as compared with control (24,197 &mgr;m2;P <0.002). Conclusions. The angiogenic growth factor bFGF can be incorporated into degradable polymers used as delivery devices for hepatocyte transplantation. Implantation of these devices increases angiogenesis into the device and increases hepatocyte engraftment.

Effect of implantation site on hepatocytes heterotopically transplanted on biodegradable polymer scaffolds.

We investigated the engraftment of heterotopically transplanted hepatocytes in three sites: the subcutaneous space, the small intestinal mesentery, and the omentum to determine the optimal location for tissue-engineered liver constructs. Hepatocytes were isolated from inbred Lewis rats and placed on polymer constructs. Cell-polymer constructs were implanted into the subcutaneous space of the abdominal wall, the small intestinal mesentery, and the omentum of Lewis rats. One group of rats had undergone previous portacaval shunt. Animals were killed 2 or 4 weeks after implantation and the constructs were analyzed for engraftment, using computer-assisted morphometric analysis. Engraftment was greatest in the omentum with less engraftment in the mesentery. There was minimal engraftment in the subcutaneous space in all specimens. Prior portacaval shunt increased engraftment in the mesentery and the omentum, but not the subcutaneous space. The omentum is the most favorable bed for engraftment of hepatocyte-polymer tissue-engineered constructs and the addition of a portacaval shunt increases survival of transplanted hepatocytes in the omentum and mesentery.

The effect of donor and recipient age on engraftment of tissue-engineered liver

A novel treatment for end-stage liver disease using heterotopic hepatocyte transplantation on biodegradable polymers has been investigated. Survival and repopulation of adequate cell mass to replace hepatic function has been the principal difficulty of this method. Hence the authors have begun to investigate the role of donor and recipient age on the efficiency of hepatocyte transplantation. Lewis rats were used as donors and recipients. Hepatocytes were isolated with a collagenase digestion, both for the adult and fetal livers (17 days estimated gestational age). After digestion, the hepatocytes were seeded onto 95% porous poly-(L)-lactic acid matrices. The polymer-cell constructs with adult or fetal cells were then implanted between mesenteric leaves of three different recipient groups: adults (approximately 200 g), 2-week, and 4-week neonates (two to five animals per group, depending on litter size). The specimens were harvested at 4 weeks, stained with Hematoxylin and Eosin (H&E), and the cell area of each specimen (24 sections per group) was quantitated using morphometric analysis. Results were statistically analyzed using an unpaired, two-tailed Students t test. At 4 weeks, all specimens showed survival of groups of hepatocytes, especially along the periphery of the polymers and near blood vessels. The hepatocyte cell area for the six groups was calculated in square micrometers: the adult cells transplanted into adult recipients, 0.16 x 10(5) microns2; fetal cells into adults, 0.47 x 10(5) microns2; adult into 4-week neonates, 1.17 x 10(5) microns2; fetal into 4-week neonates, 4.54 x 10(5) microns2; adult into 2-week neonates, 2.98 x 10(5) microns2, and fetal into 2-week neonates, 5.81 x 10(5) microns2. In all three recipient groups, the area of fetal hepatocytes was approximately two to three times the area of the adult hepatocytes (P < .05 for 2-week and 4-week neonatal recipients, P = .06 for adult recipients). Also, as the recipient age decreased, there was an increase in the hepatocyte cell area (P < .05 for fetal or adult groups). The authors conclude that fetal hepatocytes heterotopically transplanted have a significant survival advantage over adult hepatocytes, independent of recipient age. The authors further conclude that the neonatal environment is more favorable than the adult environment for implantation of hepatocytes.

A valved hepatic portoduodenal intestinal conduit for biliary atresia

Forty-six consecutive patients with biliary atresia were operated on at our institution during the 11-year period between 1978 and 1989. Their ages at operation ranged from 18 to 153 days (mean, 59 days). After dissecting the porta hepatis structures by Kasai operation, a biliointestinal anastomosis was constructed with a valved hepatic portoduodenal intestinal conduit in all cases. The intestinal valve is an intussuscepted muscular valve. Valvular function was examined radiologically. The upper gastrointestinal series demonstrated no reflux of contrast material into the conduit proximal to the valve and liver scintigraphy demonstrated that radioactive isotope drained readily into the duodenum through the valve. Thirty-nine of the forty-six patients (85%) had bile drainage after initial operation. At present 30 patients (65%) are alive without jaundice, 6 (13%) are alive with jaundice, and 10 (22%) are dead. The 5-year jaundice-free survival rate was 64%. Cholangitis occurred in 9 of 39 patients (23%) who had obtained apparent bile drainage: 5 had tractable cholangitis and 4 had refractory cholangitis. Reoperation restored bile flow in 2 of 8 patients who abruptly ceased to have bile drainage without cholangitis. In conclusion, with a valved hepatic portoduodenal intestinal conduit, the incidence of cholangitis was decreased, its medical control became easier, and the survival rate was improved.

Sclerosing therapy with bleomycin emulsion for lymphangioma in children

Forty-seven children with lymphangioma were treated by sclerosing therapy with bleomycin (BLM) emulsion between may 1976 and March 1988. Injection of 0.3 to 0.6 mg/kg body weight BLM emulsion into the cavity was repeated at intervals of 4 to 6 weeks. The lymphangioma regressed in 41 (87%) and almost disappeared in 20 (43%). The response was greater when the lymphangioma was cystic rather than cavernous. In the cases that responded, the total injected dose was 0.6 to 4 mg/kg. This therapy proved to be dangerous for large cervicomediastinal lymphangiomas in young infants because the cystic mass enlarged transiently after local injection and airway compression resulted. Pulmonary fibrosis was not observed. There was no recurrence of the residual induration after BLM emulsion therapy. Antenatal ultrasonography and magnetic resonance imaging have been shown to be useful diagnostic aids.

Alterations in Hepatic Mitochondrial Function during Total Parenteral Nutrition in Immature Rats

To evaluate the effects of total parenteral nutrition (TPN) on hepatic mitochondrial function in immature rats, changes in hepatic energy charge levels and oxidative phosphorylation rates of hepatic mitochondria were studied along with the examination of serum chemical test. Male Wistar rats weighing 30 to 45 g were used and randomized into TPN (n = 8), enteral (n = 7), and control groups (n = 8). Parenteral and enteral groups were fed with TPN solution containing 19.3% dextrose, 3.19% amino acids, 1.05% fat emulsion, minerals and vitamins, and the control group with rat chow. The number of calories per kilogram per day was 550 x 1/4 on the 1st day, 550 x 1/2 on the 2nd, 550 x 3/4 on the 3rd, and 550 x 1 on the 4th day, based on the body weight on the 1st day. After the 5th day, 550 Kcal/kg/day was given, based on the body weight of the respective day. After 13-day feeding, hepatic energy charge (EC), phosphorylation rate (PR) of hepatic mitochondria and serum chemical examination were carried out. EC was 0.871 +/- 0.016 in the control group, 0.830 +/- 0.019 in the enteral, and 0.785 +/- 0.011 in TPN group (p less than 0.001, compared with control group). PR was 138.9 +/- 1.9, 133.0 +/- 6.7, 111.0 +/- 4.3, respectively, (p less than 0.05, compared with control and enteral groups). There was no difference between the three groups on SGOT, SGPT, and total bilirubin. TPN group showed a deterioration of hepatic phosphorylation rate and energy charge in spite of normal serum transaminase levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunological Treatment with Low Dosage Ciclosporin in Rat Liver Allotransplantation

Ciclosporin (CsA) was administered subcutaneously at a dose of 3 mg/kg body weight/day from the day of operation to 14 days of liver allotransplantation in ACI rat (RT1a) to LEW rat (RT1(l) strain combination. All LEW recipients of ACI liver transplants without immunosuppressive treatment had severe rejection and expired within 12 days. In contrast, 7 out of 9 recipients in the same strain combination with temporary CsA treatment survived indefinitely. Histologically, widespread cellular infiltration and massive hepatocyte necrosis were evident upon autopsy of the recipients without CsA treatment. In contrast, in the surviving rats of the CsA-treated group, mononuclear cell infiltration was restricted to the periportal field and hepatocytes appeared to be normal at 14 days posttransplant. CsA concentrations in whole blood were determined by high-performance liquid chromatography. The trough levels were 788 +/- 48, 621 +/- 76 and 546 +/- 52 ng/ml, at 5, 10 and 14 days posttransplant, respectively. We concluded that this relatively low-dose subcutaneous administration of CsA offered adequate immunosuppression in rat liver allotransplantation in this strain combination.

Pancreaticoduodenectomy following total gastrectomy: A case report and literature review

We present a case of afferent loop syndrome (ALS) occurring after pancreaticoduodenectomy (PD) in a patient who had previously undergone total gastrectomy (TG), and review the English-language literature concerning reconstruction procedures following PD in patients who had undergone TG. The patient was a 69-year-old man who had undergone TG reconstruction by a Roux-en-Y method at age 58 years. The patient underwent PD for pancreas head adenocarcinoma. A jejunal limb previously made at the prior TG was used for pancreaticojejunostomy and hepaticojejunostomy. Despite normal patency of the hepaticojejunostomy, he suffered from repeated postoperative cholangitis which was brought on by ALS due to shortness of the jejunal limb (15 cm in length). We therefore performed receliotomy in which the hepaticojejunostomy was disconnected and reconstructed using a new Y limb 40-cm in length constructed in a double Roux-en-Y fashion. The refractory cholangitis resolved immediately after the receliotomy and did not recur. Review of the literature revealed the lack of any current consensus for a standard procedure for reconstruction following PD in patients who had previously undergone TG. This issue warrants further attention, particularly given the expected future increase in the number of PDs in patients with a history of gastric cancer.

A case of successful surgical repair for solid segment type pyloric atresia using a novel gastroduodenostomy procedure.

We present a case of successful surgical repair for solid segment type pyloric atresia using a novel gastroduodenal procedure.

Eosinophilic enteritis due to cow's milk allergy: Possible cause of anastomosis failure following repair of focal intestinal perforation.

Reports of cows milk allergy (CMA) after neonatal gastrointestinal surgery have recently increased. In recent years it has been suggested that the development of CMA after gastrointestinal surgery in newborn infants is due to an immune function. In addition, the development of CMA might be synergistically exacerbated by congenital abnormalities of the intestinal mucosa, general conditional changes and local damage to the intestine by invasive surgery, and poor pre‐ or post‐surgical nutrition. CMA manifests as a variety of symptoms, such as mild vomiting and bloody stool, decreased activity, poor oral intake, and ileus. CMA may also rarely cause gastrointestinal perforation. Here, we report the case of a newborn infant who developed CMA following repair of focal small intestinal perforation, in which eosinophilic enteritis was suspected to be a possible cause of anastomosis leakage.