Delivery of whole liver-equivalent hepatocyte mass using polymer devices and hepatotrophic stimulation.

Using hepatocytes injected into prevascularized polymer sponge devices, we studied hepatocyte survival and function after delivery of a whole liver-equivalent of cells into rats. LEW rats and enzyme-deficient Gunn rats served as recipients, respectively. Totally, 28.5 cm2 (0.5-cm thick) of polyvinyl alcohol sponges were implanted per animal. Hepatotrophic stimulation was induced by portacaval shunt and partial (70% or 30%) hepatectomy. Recipient rats received 5 x 10(8) hepatocytes (equivalent to whole rat liver) which were harvested from LEW and Wistar donors, respectively. After engraftment, histologic examination revealed hepatocyte remodeling in the device with capillaries lining plates of hepatocytes, and also tubular structures resembling early biliary radicles. BrdU staining revealed DNA synthesis in hepatocytes, providing evidence of regeneration within the grafts. Quantification of viable hepatocyte area at various time points was performed using computer-assisted morphometry. We then estimated a range of cell numbers from the quantitated cell area. The number of hepatocytes viable at day 7 was estimated at 27.5-46.0% and 6.6-11.0% in the mesentery and subcutaneous site, respectively. Thus the average number was estimated between 10.8% and 18.0% of initially injected hepatocytes. In the Gunn rat experiment, experimental rats that received normal Wistar hepatocytes showed a significantly greater decrease in total serum bilirubin compared with the concurrent control Gunn rats (P < 0.01). At week 1, serum bilirubin in experimental rats decreased to 74.7% (6.80 +/- 0.46 mg/dl) of pretransplantation level (9.10 +/- 0.47 mg/dl) and this was 71.4% of the control rats' bilirubin level (9.53 +/- 0.37 mg/dl). In conclusion, a hepatocyte mass equivalent to a whole rat liver can be delivered into prevascularized polymer sponge devices. At day 7 between 10.8% and 18.0% of these hepatocytes were estimated to be engrafted and functioning. Further optimization of this technique is necessary before clinical application is considered.

Effects of hypoxemia on early postoperative course of liver transplantation in pediatric patients with intrapulmonary shunting.

Nine pediatric patients (mean age, 10 years) with biliary atresia, who had hypoxemia related to intrapulmonary shunting, underwent living related liver transplantation. The effects of hypoxemia during the early postoperative period after liver transplantation on cardiopulmonary and renal function, as well as on transplanted liver, were analyzed. Based on the degree of shunt ratio calculated by technetium-99m macroaggregated albumin scintigraphy, the nine patients were included in the moderate group (shunt ratio under 40%, n=4) or the severe group (shunt ratio over 40%, n=5). Partial pressure of arterial oxygen was maintained at normal range in the moderate group, while that in the severe group persistently had very low values (<50 mmHg), in spite of a high degree of oxygen supply. However, all patients in the severe group maintained stable cardiopulmonary vital signs, including systemic blood pressure, heart rate, respiratory rate, and cardiac index. They also demonstrated stable renal function. None of the patients died of cardiopulmonary or renal insufficiency after transplantation, but three patients died of portal vein thrombosis, sepsis, and intracranial hemorrhage (one each). The minimal adverse effect of hypoxemia on the transplanted liver was confirmed by a rapid increase of arterial ketone body ratio, low peak values (under 200 IU/L) of aspartate aminotransferase, and a steady decrease of serum total bilirubin. Four patients encountered surgical complications, including two bile leaks from the cut liver surface, two leaks from bilioenteric anastomosis, and one intestinal perforation. Six patients suffered from bacterial infections, including four wound infections, three right subphrenic abscesses, one cholangitis, and two systemic sepses. All patients in the moderate group recovered from hypoxemia, but four of five patients in the severe group have not recovered during the follow-up period between 4 and 9 months. It was concluded that the adverse effects of hypoxemia on cardiopulmonary and renal function and transplanted liver were minimal, so that patients with severe hypoxemia could tolerate the stress of liver transplantation without special management. However, the high incidence of surgical complication and infection suggested the adverse effects of hypoxemia on wound healing and resistance to bacteria infection.

Hepatocyte transplantation using biodegradable matrices in ascorbic acid-deficient rats: comparison with heterotopically transplanted liver grafts.

BACKGROUND: Hepatocyte transplantation using polymeric matrices is under investigation as an alternative therapy for metabolic liver diseases. Long-term engraftment of hepatocytes in polymers has been demonstrated. However, the metabolic activity of hepatocytes in such devices has never been assessed in direct comparison with liver grafts. METHODS: Hepatocyte and partial liver transplantation were evaluated in the scurvy-prone osteogenic disorder Shionogi rat model. Biodegradable poly glycolic acid matrices seeded with hepatocytes equivalent to 20% of the recipient's liver mass, or 20% liver grafts were heterotopically transplanted into ascorbic acid- (AsA) deficient recipients. Recipients of cell-free matrices or AsA-deficient liver grafts served as controls. Recipients were set on AsA-free diet after transplantation. Plasma AsA levels, AsA concentrations in liver and adrenal gland tissue, and body weight ratios were assessed and H&E histology was performed. RESULTS: Recipients from the control groups showed symptoms of scurvy at 1 month after cessation of AsA supply. Hepatocyte transplantation and auxiliary liver transplantation prevented symptoms of scurvy and increased plasma and tissue AsA levels and body weight ratios. AsA levels in recipients of 20% liver grafts were comparable to normal control animals. CONCLUSIONS: Hepatocytes transplanted in polymeric matrices are able to compensate for liver-based metabolic deficiencies. Hepatocyte transplantation improves plasma AsA levels in AsA-deficient recipients. However, auxiliary liver grafts are superior to hepatocyte grafts in improving metabolic parameters. Further research work is needed to increase the efficiency of liver cell transplantation with regard to a clinical application.

Soluble forms of CD95 and CD95 ligand after living related liver transplantation.

BACKGROUND: Soluble forms of CD95 and CD95 ligand (sCD95 and sCD95L, respectively) can increase in the serum of patients with some inflammatory disease. In this study, we investigated the serum levels of sCD95 and sCD95L in liver transplantation recipients. METHODS: Serum levels of sCD95 and sCD95L in living related liver transplant recipients were analyzed by ELISA and their relation to the clinical findings estimated. RESULTS: Serum samples from the recipients did not show detectable levels of sCD95L but showed significantly increased levels of sCD95. The increase of sCD95 was positively associated with that of total-bilirubin and incidence of rejection, infection, and graft ischemia. CONCLUSIONS: The present results indicate an existence of sCD95 in the recipients of living related liver transplants. The increased serum levels of sCD95 may modify the immunological situation of the recipients after transplantation or represent the ongoing graft damage.

Efficacy of traditional herbal medicines in combination with acyclovir against herpes simplex virus type 1 infection in vitro and in vivo.

Traditional herbal medicines have been safely used for the treatment of various human diseases since ancient China. We selected 10 herbal extracts with therapeutic antiherpes simplex virus type 1 (HSV-1) activity. Among these, Geum japonicum Thunb., Rhus javanica L., Syzygium aromaticum (L.) Merr. et Perry, or Terminalia chebula Retzus showed a stronger anti-HSV-1 activity in combination with acyclovir than the other herbal extracts in vitro. When acyclovir and/or a herbal extract were orally administered at doses corresponding to human use, each of the 4 combinations significantly limited the development of skin lesions and/or prolonged the mean survival times of infected mice compared with both acyclovir and the herbal extract alone (P < 0.01 or 0.05). These combinations were not toxic to mice. They reduced virus yields in the brain and skin more strongly than acyclovir alone and exhibited stronger anti-HSV-1 activity in the brain than in the skin, in contrast to acyclovir treatment by itself. Combinations of acyclovir with historically used herbal medicines showed strong combined therapeutic anti-HSV-1 activity in mice, especially reduction of virus yield in the brain.

In vitro and in vivo degradation of porous poly(DL-lactic-co-glycolic acid) foams.

This study investigated the in vitro degradation of porous poly(DL-lactic-co-glycolic acid) (PLGA) foams during a 20-week period in pH 7.4 phosphate-buffered saline (PBS) at 37 degrees C and their in vivo degradation following implantation in rat mesentery for up to 8 weeks. Three types of PLGA 85 : 15 and three types of 50 : 50 foams were fabricated using a solvent-casting, particulate-leaching technique. The two types had initial salt weight fraction of 80 and 90%, and a salt particle size of 106-150 microm, while the third type had 90% initial weight fraction of salt in the size range 0-53 microm. The porosities of the resulting foams were 0.82, 0.89, and 0.85 for PLGA 85 : 15, and 0.73, 0.87, and 0.84 for PLGA 50 : 50 foams, respectively. The corresponding median pore diameters were 30, 50, and 17 microm for PLGA 85: 15, and 19, 17, and 17 microm for PLGA 50 : 50. The in vitro and in vivo degradation kinetics of PLGA 85: 15 foams were independent of pore morphology with insignificant variation in foam weight, thickness, pore distribution, compressive creep behavior, and morphology during degradation. The in vitro foam half-lives based on the weight average molecular weight were 11.1 +/- 1.8 (80%, 106-150 microm), 12.0 +/- 2.0 (90%, 106-150 microm), and 11.6 +/- 1.3 (90%, 0-53 microm) weeks, similar to the corresponding values of 9.4 +/- 2.2, 14.3 +/- 1.5, and 13.7 +/- 3.3 weeks for in vivo degradation. In contrast, all PLGA 50 : 50 foams exhibited significant change in foam weight, water absorption, and pore distribution after 6-8 weeks of incubation with PBS. The in vitro foam half-lives were 3.3 +/- 0.3 (80%, 106-150 microm), 3.0 +/- 0.3 (90%, 106-150 microm), and 3.2 +/- 0.1 (90%, 0-53 microm) weeks, and the corresponding in vivo half-lives were 1.9 micro 0.1, 2.2 +/- 0.2, and 2.4 +/- 0.2 weeks. The significantly shorter half-lives of PLGA 50: 50 compared to 85: 15 foams indicated their faster degradation both in vitro and in vivo. In addition, PLGA 50: 50 foams exhibited significantly faster degradation in vivo as compared to in vitro conditions due to an autocatalytic effect of the accumulated acidic degradation products in the medium surrounding the implants. These results suggest that the polymer composition and environmental conditions have significant effects on the degradation rate of porous PLGA foams.

Hepatocyte transplantation in Swine using prevascularized polyvinyl alcohol sponges.

Hepatocyte transplantation shows promise as therapy to support liver function. We have shown that hepatocytes can be transplanted into prevascularized synthetic polymers in rat models. While there are many studies in rats showing the benefits of hepatocyte transplantation, there are few in larger animal models more akin to humans. Therefore, we developed a swine model of hepatocyte transplantation using prevascularized synthetic polymers. Polyvinyl alcohol sponges measuring 2 x 3 x 0.5 cm were implanted in preperitoneal, mesenteric, and subcutaneous spaces for prevascularization as a transplantation bed. On postimplantation day 0, 4, 8, or 12 the sponges were removed and examined histologically. New tissue ingrowth was quite satisfactory on day 8 and 12 in preperitoneal and mesenteric sites, but the sponges in subcutaneous tissue were compressed, leaving little space for hepatocyte engraftment. Mesenteric sponges were irritating to intraabdominal organs and induced peritoneal adhesions. Thus, the preperitoneal sponges seemed to be best for hepatocyte transplantation. Hepatocytes isolated from donor livers using collagenase perfusion were transplanted into the preperitoneal sponges prevascularized for 0, 4, 8, or 12 days as allografts with cyclosporine immunosuppression, and the number of hepatocytes was evaluated on posttransplantation day 4. The optimal period for prevascularization for subsequent hepatocyte implantation was 8 days. The number of hepatocytes implanted in the preperitoneal sponges prevascularized for 8 days was counted on day 0, 1, 4, or 8 after transplantation. Hepatocytes were lost mainly in the first day after implantation, but still many hepatocytes maintained their shape histologically and there were mitotic figures confirming growth of the transplanted hepatocytes. Areas of hepatocytes within the sponge devices showed tissue remodeling with plates of hepatocytes lined with sinusoid-like capillaries and evidence of early tubular formation. Positive staining by immunohistochemical examination using antipig albumin indicated albumin production by implanted hepatocytes. The effect of a portacaval shunt as a hepatotrophic stimulation to maintenance of the implanted hepatocytes was evaluated on day 4, 8, or 12 after implantation. Total hepatocyte number in sponges was significantly increased by portacaval shunt compared to controls treated by a sham operation. These results suggest that significant numbers of hepatocytes can engraft and function using a prevascularized polymer bed as a site for transplantation and ongoing hepatotrophic stimulation with portacaval shunting.

Kinetic analysis of the preserved rat liver by isolated perfusion with ammonium chloride as a load.

Oxygen consumption and urea synthesis from ammonium chloride (NH4Cl) were investigated in the liver preserved in University of Wisconsin solution at 4 degrees C for 24 hours using an isolated rat liver perfusion system in which the perfusate contained five different concentrations of NH4Cl. When a Michaelis-Menten equation was applied to oxygen consumption and urea synthesis against NH4Cl concentration, the preserved liver showed smaller increase in oxygen consumption rate and larger Km of urea synthesis for NH4Cl than the fresh liver. The ratio of respiration velocity without any substrate to maximal velocity (v/Vmax), which reflects the mitochondrial functional reserve, was 55.9 +/- 4.1% and 41.5 +/- 4.8% in the preserved and fresh liver, respectively (p less than 0.05). From the viewpoint of work-cost relationship, it was shown that the mitochondrial function in the preserved liver was deteriorated. On the other hand, conventional mitochondrial study after rewarming and reoxygenation but before NH4Cl load revealed no deterioration of mitochondrial function after preservation. These results indicate that it is necessary to take the metabolic load on the reperfused liver into account when assessing graft viability, and that high v/Vmax suggests decrease in the reserve of mitochondrial function under consideration of the metabolic load.

Human hepatocyte isolation and transplantation into an athymic rat, using prevascularized cell polymer constructs.

Human hepatocyte viability and function in vivo in an athymic rat was assessed after transplantation on prevascularized polymer constructs with hepatotrophic stimulation. Sixteen liver biopsy specimens, weighing 5 to 12 g, were obtained from the New England Organ Bank and from the operating room after liver resection. In the laboratory they were catheterized and perfused to obtain liver cell suspensions. From eight of the 16 cell suspensions, only in vitro studies were performed. They showed 40% cell attachment 24 hours after initial cell plating. For patients aged 2, 35, and 60 years, they showed a 20% increase, a 1% decrease, and a 57% decrease (respectively) in cell number from day 2 to day 4, after cell plating. Eight cell suspensions were transplanted into athymic rats. On sections examined histologically, implanted hepatocytes were seen within the fibroblast ingrowth, in the space of the polymer device, until day 21 after cell injection. On day 9 after hepatocyte injection, reorganized hepatic parenchyma was seen on the tissue section. Implanted hepatocyte areas, quantitated through morphometric analysis on days 0, 3, and 7, showed a 36% increase in engraftment 3 days after injection, and a 42% decrease 7 days after injection. At the same time-points, immunoperoxidase staining visualized intracellular albumin, which was specific for the implanted hepatocytes. In conclusion, the authors demonstrated the feasibility of their technique (prevascularized polymer device with hepatotrophic stimulation), using human hepatocytes. Further studies are underway, before implementation of human clinical trials.

Evaluation of methods of hepatotrophic stimulation in rat heterotopic hepatocyte transplantation using polymers.

BACKGROUND: Hepatocyte transplantation has been studied as an alternative to organ transplantation. Hepatocyte transplant models should provide sufficient cell mass for replacement function and hepatotrophic stimulation of the transplanted cells in heterotopic locations. METHOD: The authors used three-dimensional porous polyvinyl-alcohol matrices as cell carriers, which were implanted between mesenteric leaves of the intestine. In this study, different methods were evaluated for hepatotrophic stimulation. Fifty million transplanted hepatocytes (approximately 10% liver mass) were implanted in Lewis rats. We compared 70% partial hepatectomy, portacaval shunt, cotransplantation of enterocytes, cotransplantation of islets of Langerhans, and methylprednisolone injection to a control group with only hepatocyte transplantation. Portacaval shunt and islet cotransplantation also were used in combination. Specimens were harvested 2 weeks after transplantation, and area per histological cross section compromised by hepatocytes was measured. RESULTS: Seventy percent partial hepatectomy, enterocyte cotransplantation, and methylprednisolone injection resulted in hepatocyte maintenance similar to control group (3,100 +/- 7,592 microm2). Portacaval shunt (96,866 +/- 55,039 microm2) and islet cotransplantation (173,020 +/- 75,977 microm2) yielded a highly significant increase in hepatocyte area. The combination of portacaval shunt and islet cotransplantation resulted in a significant increase compared with using these methods individually (288,930 +/- 86,726 microm2). Additional immunohistochemical stains for active DNA synthesis, insulin, and glucagon demonstrated the proliferative abilities of the hepatocytes and the synthesis of insulin and glucagon in the cotransplanted islets. CONCLUSION: Hepatocyte transplantation can be performed using polymer carriers and that hepatocyte survival and maintenance can be improved with portacaval shunt and islet cotransplantation.

Low-temperature fluorometric technique for evaluating the viability of rat liver grafts after simple cold storage.

Time-dependent changes in the viability of rat liver graft during cold preservation with Euro-Collins solution were evaluated with NADH fluorometry. Correlation between the fluorometric analysis, 1-week survival rate after liver transplantation, and mitochondrial ATP synthesis activity in the early phase after transplantation was studied. Fluorometric study: Rat livers were preserved at 0 degree-4 degrees C for 0-48 h in Euro-Collins solution and then reperfused for 15 min with oxygenated Krebs-Henseleit solution at 4 degrees C. The amplitude (R x A) between the oxidized and the reduced steady-state NADH fluorometric trace and the velocity (R x V) of the trace were determined to evaluate the mitochondrial respiratory chain. The R x A and R x V remained at levels higher than 90% of control after 6-h preservation, while the R x A of the 9-h preservation group and the R x V of the 12-h preservation group decreased significantly compared with those of the control and the 6-h preservation group. Survival study: a 100% survival rate after transplantation was achieved in the 6-h preservation group, whereas the rates were 18.8% and 0% in the 9- and 12-h preservation groups respectively. These survival rates correlated closely with the time-dependent decrease of the fluorometric parameters. Study of mitochondrial phosphorylative activity and energy charge 3 h after transplantation: With fresh grafts, the decrease in hepatic energy charge after transplantation was reduced to 0.79 from the control value of 0.86 by a 30% increase in mitochondrial ATP synthesis ability.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of enhanced mitochondrial phosphorylation in rat liver transplantation.

The effects of organ preservation on mitochondrial oxidative phosphorylation activity, adenylate hepatic energy charge, cytochrome content, and redox state of NAD+/NADH couple in rat liver transplantation were compared between a nonpreservation group and a preservation group with grafts preserved for 12 hr in Euro-Collin's solution. At 3 hr after transplantation, the energy charge in the preservation group decreased to 0.60 +/- 0.02 from the control value of 0.86 +/- 0.01, accompanied by a reduction of intramitochondrial redox state of NAD+/NADH couple. In contrast, in the nonpreservation group, the decrease in energy charge was minimally decreased to 0.79 +/- 0.04 due to the compensatory enhancement of mitochondrial oxidative phosphorylation activity. These results suggest that an enhanced mitochondrial ATP synthesis and a reduced intramitochondrial redox state are important factors affecting survival following rat liver transplantation.

Application of a tissue expander for stabilizing graft position in living-related liver transplantation.

In living-related liver transplantation, a portion of the whole liver, usually the left lateral segment or the left lobe, is harvested for use as the graft liver. A relatively small graft may twist around the hepatic venous anastomosis, causing outflow blockage. After an initial experience of such a complication in the early post-transplantation period in one case, we began to apply a tissue expander to obliterate the dead space left after emplacement of a relatively small graft. Commercially available tissue expanders, ordinary employed in plastic surgery, were used for five pediatric recipients (6-14 yr old) whose ratio of graft weight to recipient body weight ranged from 0.74 to 1.72. The expander, filled with 240-350 ml saline, was put into the space to the right of the graft. Aspiration of the expander content was started after the 5th-7th postoperative day (POD), at a rate of 50 ml of fluid a day. The expanders were taken out on the 11th-14th POD after complete removal of the content. Complications observed in the five cases were right shoulder pain (5/5), fluid collection after removal of the expander(2/5), chylous ascites (1/5), and one instance of bile leakage. All the complications were successfully managed and controlled conservatively. There was no outflow obstruction detected by Doppler ultrasonography before or after removal of the expander. This is a simple and safe method for preventing the early onset of outflow block after partial liver transplantation.