A Novel Approach to Orthotopic Hepatocyte Transplantation Engineered With Liver Hydrogel for Fibrotic Livers, Enhancing Cell-Cell Interaction and Angiogenesis.

Hepatocyte transplantation (HCT) is a potential bridging therapy or an alternative to liver transplantation. Conventionally, single-cell hepatocytes are injected via the portal vein. This strategy, however, has yet to overcome poor cell engraftment and function. Therefore, we developed an orthotopic HCT method using a liver-derived extracellular matrix (L-ECM) gel. PXB cells (flesh mature human hepatocytes) were dispersed into the hydrogel solution in vitro, and the gel solution was immediately gelated in 37°C incubators to investigate the affinity between mature human hepatocyte and the L-ECM gel. During the 3-day cultivation in hepatocyte medium, PXB cells formed cell aggregates via cell-cell interactions. Quantitative analysis revealed human albumin production in culture supernatants. For the in vivo assay, PXB cells were encapsulated in the L-ECM gel and transplanted between the liver lobes of normal rats. Pathologically, the L-ECM gel was localized at the transplant site and retained PXB cells. Cell survival and hepatic function marker expression were verified in another rat model wherein thioacetamide was administered to induce liver fibrosis. Moreover, cell-cell interactions and angiogenesis were enhanced in the L-ECM gel compared with that in the collagen gel. Our results indicate that L-ECM gels can help engraft transplanted hepatocytes and express hepatic function as a scaffold for cell transplantation.

Novel approach for reconstruction of the three-dimensional biliary system in decellularized liver scaffold using hepatocyte progenitors.

Reconstruction of the biliary system is indispensable for the regeneration of transplantable liver grafts. Here, we report the establishment of the first continuous three-dimensional biliary system scaffold for bile acid excretion using a novel method. We confirmed the preservation of the liver-derived extracellular matrix distribution in the scaffold. In addition, hepatocyte progenitors decellularized via the bile duct by slow-speed perfusion differentiated into hepatocyte- and cholangiocyte-like cells, mimicking hepatic cords and bile ducts, respectively. Furthermore, qRT-PCR demonstrated increased ALB, BSEP, and AQP8 expression, revealing bile canaliculi- and bile duct-specific genetic patterns. Therefore, we concluded that locally preserved extracellular matrices in the scaffold stimulated hepatic progenitors and provided efficient differentiation, as well as regeneration of a three-dimensional continuous biliary system from hepatic cords through bile ducts. These findings suggest that organ-derived scaffolds can be utilized for the efficient reconstruction of functional biliary systems.

A thioacetamide-induced liver fibrosis model for pre-clinical studies in microminipig.

Drug-induced liver fibrosis models are used in normal and immunosuppressed small animals for transplantation and regenerative medicine to improve liver fibrosis. Although large animal models are needed for pre-clinical studies, they are yet to be established owing to drug sensitivity in animal species and difficulty in setting doses. In this study, we evaluated liver fibrosis by administering thioacetamide (TA) to normal microminipig and thymectomized microminipig; 3 times for 1 week (total duration: 8 weeks). The pigs treated with TA showed elevated blood cytokine levels and a continuous liver injury at 8 weeks. RNA-seq of the liver showed increased expression of fibrosis-related genes after TA treatment. Histopathological examination showed degenerative necrosis of hepatocytes around the central vein, and revealed fibrogenesis and hepatocyte proliferation. TA treatment caused CD3-positive T cells and macrophages scattered within the hepatic lobule to congregate near the center of the lobule and increased αSMA-positive cells. Thymectomized pigs showed liver fibrosis similar to that of normal pigs, although the clinical signs tended to be milder. This model is similar to pathogenesis of liver fibrosis reported in other animal models. Therefore, it is expected to contribute to research as a drug discovery and pre-clinical transplantation models.

Effects of a Soluble Guanylate Cyclase Stimulator Riociguat on Contractility of Isolated Pulmonary Artery and Hemodynamics of U46619-Induced Pulmonary Hypertension in Dogs.

Soluble guanylate cyclase (sGC) stimulator riociguat is a relatively novel therapeutic agent for pulmonary hypertension (PH) in human medicine. Riociguat induces endothelium-independent pulmonary artery (PA) relaxation by directly activating the sGC-cyclic guanosine-3',5'-monophosphate (cGMP) pathway in muscle cells. Although riociguat may be effective in the treatment of dogs with refractory PH, basic studies on its clinical application in veterinary medicine are lacking. The present study aimed to explore the effects of riociguat on the contractility of an isolated canine PA and the hemodynamics of dogs with acute PH. In an isolated endothelium-denuded canine PA, the effects of riociguat on endothelin (ET)-1-induced contraction and cGMP levels were investigated using the Magnus method and ELISA, respectively. The effect of riociguat on the hemodynamics of the thromboxane A2 analog U46619-induced PH model dog was examined by invasive catheterization. Riociguat increased cGMP levels and reduced ET-1-induced contraction of the isolated PA. Riociguat inhibited the U46619-induced elevation of PA pressure and pulmonary vascular resistance and increased cardiac output, but it had no effect on basal systemic blood pressure. These results demonstrate for the first time that riociguat can inhibit the elevation of PA pressure through PA relaxation via an endothelium-independent increase in cGMP in dogs with PH.

Decellularized Organ-Derived Scaffold Is a Promising Carrier for Human Induced Pluripotent Stem Cells-Derived Hepatocytes.

Human induced pluripotent stem cells (hiPSCs) are a promising cell source for elucidating disease pathology and therapy. The mass supply of hiPSC-derived cells is technically feasible. Carriers that can contain a large number of hiPSC-derived cells and evaluate their functions in vivo-like environments will become increasingly important for understanding disease pathogenesis or treating end-stage organ failure. hiPSC-derived hepatocyte-like cells (hiPSC-HLCs; 5 × 108) were seeded into decellularized organ-derived scaffolds under circumfusion culture. The scaffolds were implanted into immunodeficient microminiature pigs to examine their applicability in vivo. The seeded hiPSC-HLCs demonstrated increased albumin secretion and up-regulated cytochrome P450 activities compared with those in standard two-dimensional culture conditions. Moreover, they showed long-term survival accompanied by neovascularization in vivo. The decellularized organ-derived scaffold is a promising carrier for hiPSC-derived cells for ex vivo and in vivo use and is an essential platform for regenerative medicine and research.

An organ-derived extracellular matrix triggers in situ kidney regeneration in a preclinical model.

It has not been considered that nephrons regenerate in adult mammals. We present that an organ-derived extracellular matrix in situ induces nephron regeneration in a preclinical model. A porcine kidney-derived extracellular matrix was sutured onto the surface of partial nephrectomy (PN)-treated kidney. Twenty-eight days after implantation, glomeruli, vessels, and renal tubules, characteristic of nephrons, were histologically observed within the matrix. No fibrillogenesis was observed in the matrix nor the matrix-sutured kidney, although this occurred in a PN kidney without the matrix, indicating the structures were newly induced by the matrix. The expression of renal progenitor markers, including Sall1, Six2, and WT-1, within the matrix supported the induction of nephron regeneration by the matrix. Furthermore, active blood flow was observed inside the matrix using computed tomography. The matrix provides structural and functional foundations for the development of cell-free scaffolds with a remarkably low risk of immune rejection and cancerization.

Transplantation of bioengineered liver capable of extended function in a preclinical liver failure model.

Unlimited organ availability would represent a paradigm shift in transplantation. Long-term in vivo engraftment and function of scaled-up bioengineered liver grafts have not been previously reported. In this study, we describe a human-scale transplantable liver graft engineered on a porcine liver-derived scaffold. We repopulated the scaffold parenchyma with primary hepatocytes and the vascular system with endothelial cells. For in vivo functional testing, we performed auxiliary transplantation of the repopulated scaffold in pigs with induced liver failure. It was observed that the auxiliary bioengineered liver graft improved liver function for 28 days and exhibited upregulation of liver-specific genes. This study is the first of its kind to present 28 days of posttransplant evaluation of a bioengineered liver graft using a preclinical large animal model. Furthermore, it provides definitive evidence for the feasibility of engineering human-scale transplantable liver grafts for clinical applications.

A case of papillary squamous cell carcinoma in the mandible of a young French bulldog.

A 7-month-old castrated French bulldog was presented with a left-sided mandibular tumor. The initial tumor biopsy diagnosis was ameloblastoma. The owner brought this dog the Kitasato University Veterinary Teaching Hospital for more detailed examination and treatment. Computed tomography revealed a tumor on the left lateral mandibular gingiva from the caudal third of the incisor tooth to the right canine tooth, associated with severe amorphous osteolysis of the mandibular bone. The tumor was surgically excised and diagnosed as papillary squamous cell carcinoma. Currently, 2514 d (6.9 y) since the operation, the dog is healthy, without recurrence. Key clinical message: Although papillary squamous cell carcinoma is rare, many cases have been reported in the oral cavity of medium-to large-sized dogs. Based on this report, papillary squamous cell carcinoma can occur in small dogs such as young French bulldogs and a good prognosis can be achieved with proper resection.

Un cas de carcinome épidermoïde papillaire de la mandibule d'un jeune bouledogue français. Un bouledogue français castré de 7 mois a été présenté avec une tumeur mandibulaire gauche. Le diagnostic initial de biopsie tumorale était un améloblastome. Le propriétaire a amené ce chien à l'hôpital universitaire vétérinaire de Kitasato pour un examen et un traitement plus détaillés. La tomodensitométrie a révélé une tumeur de la gencive mandibulaire latérale gauche du tiers caudal de l'incisive à la canine droite, associée à une ostéolyse amorphe sévère de l'os mandibulaire. La tumeur a été excisée chirurgicalement et diagnostiquée comme un carcinome épidermoïde papillaire. Actuellement, 2514 jours (6,9 ans) depuis l'opération, le chien est en bonne santé, sans récidive.Message clinique clé :Bien que le carcinome épidermoïde papillaire soit rare, de nombreux cas ont été rapportés dans la cavité buccale de chiens de taille moyenne à grande. Sur la base de ce rapport, le carcinome épidermoïde papillaire peut survenir chez les petits chiens tels que les jeunes bouledogues français et un bon pronostic peut être obtenu avec une résection appropriée.(Traduit par Dr Serge Messier).

Timing urinary tract reconstruction in rats to avoid hydronephrosis and fibrosis in the transplanted fetal metanephros as assessed using imaging.

Chronic kidney disease leads to high morbidity rates among humans. Kidney transplantation is often necessary for severe symptoms; however, options for new curative treatments are desired because of donor shortage. For example, it has been established that the kidneys can efficiently generate urine after transplantation of the metanephros, ureter, and bladder as a group. After transplantation, the urine can indirectly flow into the recipient's bladder using a stepwise peristaltic ureter system method where the anastomosis is created via the recipient's ureter for urinary tract reconstruction. However, the growth of the regenerated metanephros varies significantly, whereas the time window for successful completion of the stepwise peristaltic ureter system that does not cause hydronephrosis of the metanephros with bladder (ureter) is quite narrow. Therefore, this study was conducted to periodically and noninvasively evaluate the growth of the transplanted metanephros, ureter, and bladder in rats through computed tomography and ultrasonography. The ultrasonographic findings highly correlated to the computed tomography findings and clearly showed the metanephros and bladder. We found that the degree of growth of the metanephros and the bladder after transplantation differed in each case. Most of the rats were ready for urinary tract reconstruction within 21 days after transplantation. Optimizing the urinary tract reconstruction using ultrasonography allowed for interventions to reduce long-term tubular dilation of the metanephros due to inhibited overdilation of the fetal bladder, thereby decreasing the fibrosis caused possibly by transforming growth factor-ß1. These results may be significantly related to the long-term maturation of the fetal metanephros and can provide new insights into the physiology of transplant regeneration of the metanephros in higher animals. Thus, this study contributes to the evidence base for the possibility of kidney regeneration in human clinical trials.

Prevention of Chronic Rejection of Marginal Kidney Graft by Using a Hydrogen Gas-Containing Preservation Solution and Adequate Immunosuppression in a Miniature Pig Model.

In clinical kidney transplantation, the marginal kidney donors are known to develop chronic allograft rejection more frequently than living kidney donors. In our previous study, we have reported that the hydrogen gas-containing organ preservation solution prevented the development of acute injuries in the kidney of the donor after cardiac death by using preclinical miniature pig model. In the present study, we verified the impact of hydrogen gas treatment in transplantation with the optimal immunosuppressive protocol based on human clinical setting by using the miniature pig model. Marginal kidney processed by hydrogen gas-containing preservation solution has been engrafted for long-term (longer than 100 days). A few cases showed chronic rejection reaction; however, most were found to be free of chronic rejection such as graft tissue fibrosis or renal vasculitis. We concluded that marginal kidney graft from donor after cardiac death is an acceptable model for chronic rejection and that if the transplantation is carried out using a strict immunosuppressive protocol, chronic rejection may be alleviated even with the marginal kidney.

Postnatal management of prenatally diagnosed biliary cystic malformation.

PURPOSE: Recent advances in ultrasonography have increased prenatal diagnosis of biliary atresia (BA) and choledochal cyst (CC). These conditions are not easy to distinguish before or just after birth. This study investigated diagnostic and therapeutic problems in prenatal diagnosis of BA and CC. METHODS: We retrospectively studied clinical characteristics and progression of hepatobiliary cysts in 10 patients (4 cases of BA, 6 cases of CC) from the time of diagnosis. Chronological changes in cyst size and gallbladder morphology were assessed and measured sequentially by ultrasonography. RESULTS: Three cases of BA were type I cyst and 1 case was type III-d. All cases of CC were type Ia. Cyst size decreased between birth and surgery in BA but increased in CC. The gallbladder appeared atrophic in BA. There was no significant difference in gestational age or cyst size at prenatal diagnosis, changes in cyst size between birth and surgery, and degree of liver fibrosis. CONCLUSIONS: BA should be suspected if cyst size decreases before and after birth and the gallbladder atrophies after birth. Cholangiography is the only reliable method to differentiate BA from CC. Neonatal surgery is indicated for CC with icterus and liver dysfunction.

Liver transplantation following the Kasai procedure in treatment of biliary atresia: a single institution analysis.

PURPOSE: This study aimed to assess outcomes of liver transplantation (LTx) in patients with biliary atresia (BA). METHODS: The Kasai procedure was performed for 358 patients at Tohoku University Hospital between January 1955 and December 2013; 64 (17.9%) required LTx. These 64 patients were divided into 4 groups according to their age at the time of transplantation: Group 1, aged <2 years (n = 27); Group 2, aged 2-9 years (n = 16); Group 3, aged 10-19 years (n = 11); and Group 4, aged ≥20 years (n = 10). Clinical parameters were evaluated retrospectively. RESULTS: Both living-donor (n = 57) and deceased-donor (n = 7) LTx were performed. Indications were irreversible jaundice (n = 53), intractable cholangitis (n = 3), hepatopulmonary syndrome (n = 6), portopulmonary hypertension (n = 1), and intestinal bleeding (n = 1). Jaundice occurred more frequently in Groups 1 and 2 than in Groups 3 and 4 (p = 0.031). Survival rates were 81.5, 100, 90.9, and 80% in Groups 1, 2, 3, and 4, respectively. CONCLUSION: Although the overall LTx survival rate was satisfactory, some adult recipients experienced LTx-related difficulty. Close follow-up, meticulous assessment of physical and social conditions, presence of a multidisciplinary support system, and appropriate time course for LTx are all essential factors in the treatment of BA.

Life-threatening risk factors and the role of intestinal transplantation in patients with intestinal failure.

PURPOSE: We elucidated the life-threatening risk factors for intestinal failure (IF) and characterized the role of intestinal transplantation (ITx) in affected patients. METHODS: We conducted a retrospective review of 38 patients with short bowel (SB) and 19 with motility disorders (MD). The SB patients were divided into three categories according to the length of their residual small bowel and the presence of the ileocecal valve. The four disease subcategories were grouped into two categories: low-risk category (mild and moderated SB) and high-risk category (extensive SB and MD). The age at the introduction of parenteral nutrition (PN) was <1 year in 50 patients (infant group, IG) and 1-15 years in 7 patients (pediatric group, PG). RESULTS: Enteral autonomy was rarely achieved in the high-risk category (p < 0.0001). IG was associated with a higher incidence of developing intestinal failure-associated liver disease (IFALD) (p = 0.004). Eight patients died, due to IFALD in four, sepsis in three and acute heart failure in one. Twenty-eight patients (49 %) are currently alive without PN, including four after ITx. CONCLUSION: The treatment of high-risk IF is still challenging. Inclusion of ITx in appropriate timing, along with aggressive medical, nutritional and surgical management, may reduce advanced morbidity and mortality of high-risk IF.

Impact of age at Kasai operation on short- and long-term outcomes of type III biliary atresia at a single institution.

PURPOSE: We reevaluated the impact of age at Kasai operation on the short- and long-term outcomes of type III biliary atresia (BA). PATIENTS AND METHODS: From 1953 to 2009, 242 patients with type III BA underwent Kasai operation at ages ranging between 12 and 421 days (average, 79.7 days). The relationship between age at Kasai operation and jaundice disappearance rates (JDRs), and 10-, 20-, and 30-year native liver survival rates (NLSRs) were assessed retrospectively (JDR [%] = the number of patients in whom jaundice disappeared/the number of patients in each group × 100). RESULTS: Age at Kasai operation had a significant impact on the JDRs (P < .001). However, there was no statistical relationship between long-term NLSR of the patients in whom jaundice disappeared after Kasai operation and operative age. From the results of the cumulative NLSRs estimated by Kaplan-Meier method, each survival rate was quite dependent on the age at operation until 30 years after Kasai operation, but the difference became much smaller in the later period provided age at operation was 4 months or younger. CONCLUSION: The operative age as a prognostic factor might be less significant in the long-term outcome than in the short-term outcome.

[Complications in long-term survivors of biliary atresia].

While most long-term survivors of biliary atresia lead lives of good quality, some patients experience late complications. Cholangitis is a common complication even among 20-year survivors including some who develop ongoing liver dysfunction. Portal hypertension is another late complication. Endoscopic treatment for esophageal varices and partial splenic embolization for hypersplenism is effective as long as hepatic functional reserve is preserved. On the other hand, the treatment of secondary intrapulmonary vascular disorders including hepatopulmonary syndrome and portopulmonary hypertension is difficult. Early liver transplantation is recommended for these conditions. Pregnancy and delivery can be stressful events potentially leading to liver failure in female patients. Even very long-term survivors should be carefully monitored in terms of liver function, portal hypertension, and cholangitis.

Living-related intestinal transplantation for a patient with hypoganglionosis.

A 14-yr-old boy with total parenteral nutrition-dependent short-bowel syndrome associated with hypoganglionosis underwent the LR-IT by using a 150 cm segment of distal ileum taken from a healthy donor. The graft vessels were connected to infrarenal aorta and inferior vena cava. The immunosuppressive regimen consisted of daclizumab, tacrolimus, and steroid. The graft surveillance for ACR was accomplished using zoom endoscopy and mucosal biopsy. The blood trough level of tacrolimus was maintained between 20 and 25 ng/mL for the first 2 months, followed by 15-20 ng/mL thereafter. The 50 mg of daclizumab was administered on the day of operation, and same dosage was repeated at 2-wk intervals. The first ACR occurred on POD-9 and was progressive, and required a 14-day course of OKT-3 injection. After the treatment with OKT-3, the graft recovered from the ACR, and began to function well enough to discontinue the intravenous nutrition on POD-55. No infectious complication has occurred. The patient was discharged in POD-112, and currently tolerates full oral intake without requiring intravenous nutritional or fluid supplementation. The donor was discharged without any complications. The LR-IT could successfully be performed with minimal risk to the donor, and it can be a treatment of choice for patients with short-gut syndrome associated with hypoganglionosis.

An experimental study on the utility of a 3-mm ultrasonically activated trocar system.

BACKGROUND/PURPOSE: A prototype of a 3-mm ultrasonically activated trocar system supplied by Olympus Corporation was evaluated experimentally in terms of its utility and safety. METHODS: Three piglets with an average weight of 12 kg were used. A pneumoperitoneum was created by Hasson's technique. Eleven punctures were made with a disposable conical 3-mm trocar (CT), 9 punctures with a 3-mm radially expanding trocar (ET), and 13 punctures with a 3-mm ultrasonically activated trocar (UT) under laparoscopic control. The authors recorded the time for abdominal penetration, the severity of the peritoneal tenting, the presence of elevated abdominal pressure of 5 mm Hg or more at the penetration, and the maximal force applied to the trocar to remove from the abdominal wall. RESULTS: The average times for penetration were 11.8, 9.4, and 3.8 seconds with CT, ET, and UT, respectively (P < .05, CT v. UT, ET v. UT). The average maximal forces at the trocar removal were 10.52, 21.17, and 21.24 N with CT, ET, and UT, respectively (P < .05, CT v. ET, CT v. UT). Elevation of abdominal pressure of 5 mm Hg or more was recorded in CT and ET but not in UT. Peritoneal tenting was the most severe in ET and minimal in UT. No complication related to the UT system was found. CONCLUSIONS: The 3-mm UT is a simple and safe device and is expected to become commercially available.

Functional evaluation of an artificial anal sphincter using shape memory alloys.

This article describes an implantable artificial anal sphincter using shape memory alloys and its in vivo assessment in porcine models. The new design was developed as a low invasive prosthesis with a simple structure to solve the problem of severe fecal incontinence in patients with hypoplastic sphincters or without anal sphincters and especially for ostomates. The artificial anal sphincter consists of two shape memory alloy (SMA) plates as the main functional parts to perform two basic functions when the SMA artificial sphincter is fitted around intestines (i.e., an occlusion at body temperature and an opening function on heating). Our previous assessments with short-term animal experiments revealed promising properties with the occlusion function of the device, although some complications, such as overpressure induced ischemia, heat burn, and infections, remained. This article addresses the concerns related to the practical use of the device, the power supplement to drive the actuator, and overheating protection of the device inside bodies. Results of chronic animal experiments of up to 4 weeks suggested great potential for the improved device.

Development of an implantable artificial anal sphincter using a shape memory alloy.

BACKGROUND/PURPOSE: The authors created a new artificial anal sphincter using a shape memory alloy (AS-SMA) to treat fecal incontinence and evaluated its validity. METHODS: AS-SMA consists of 2 Ti-Ni plates to sandwich the intestine and generates a pressure of 55 mm Hg at its resting position. With the electric power supply, the 2 metals bend to form an almondlike shape making a maximum gap of 33 mm between each other at the temperature of 55 degrees C. The device was attached to the colostomy in a piglet and was operated several times a day for 1 month. Fecal continence, bowel movements, and general condition of the piglet were recorded. After the experiment, tissue damage around the device was investigated. RESULTS: Fecal continence was obtained while the device was in the resting position. When it was operated, bowel movements were observed. The bowel movements to operations ratio was 82:105 (78%). During the experiment, the animal had neither abdominal distension nor vomiting. At the autopsy, there was mild inflammation and shallow burns around the device. No compression injury was detected. CONCLUSIONS: AS-SMA achieved fecal continence of the colostomy. With reduction of the associated side effects, it would be a potential substitute for an impaired anal sphincter.