Amedeo Carraro

Graphene based scaffolds effects on stem cells commitment

Graphene is a flat monolayer of carbon atoms, arranged in a two-dimensional hexagonal structure, with extraordinary electrical, thermal, and physical properties. Moreover, the molecular structure of graphene can be chemically modified with molecules of interest to promote the development of high-performance devices. Although carbon derivatives have been extensively employed in industry and electronics, their use in regenerative medicine is still in an early phase. Study prove that graphene is highly biocompatible, has low toxicity and a large dosage loading capacity. This review describes the ability of graphene and its related materials to induce stem cells differentiation into osteogenic, neuronal, and adipogenic lineages.

Systemic administration of a novel human umbilical cord mesenchymal stem cells population accelerates the resolution of acute liver injury

BackgroundHepatocytes and stem cells transplantation may be an alternative to liver transplantation in acute or chronic liver disease. We aimed to evaluate the therapeutic potential of mesenchymal stem cells from human umbilical cord (UCMSCs), a readily available source of mesenchymal stem cells, in the CCl4-induced acute liver injury model.MethodsMesenchymal stem cells profile was analyzed by flow cytometry. In order to evaluate the capability of our UCMSCs to differentiate in hepatocytes, cells were seeded on three different supports, untreated plastic support, MatrigelTM and human liver acellular matrix. Cells were analyzed by immunocitochemistry for alpha-fetoprotein and albumin expression, qPCR for hepatocyte markers gene expression, Periodic Acid-Schiff staining for glycogen storage, ELISA for albumin detection and colorimetric assay for urea secretion.To assess the effects of undifferentiated UCMSCs in hepatic regeneration after an acute liver injury, we transplanted them via tail vein in mice injected intraperitoneally with a single dose of CCl4. Livers were analyzed by histological evaluation for damage quantification, immunostaining for Kupffer and stellate cells/liver myofibroblasts activation and for UCMSCs homing. Pro- and anti-inflammatory cytokines gene expression was evaluated by qPCR analysis and antioxidant enzyme activity was measured by catalase quantification.Data were analyzed by Mann–Whitney U-test, Kruskal-Wallis test and Cuzick’s test followed by Bonferroni correction for multiple comparisons.ResultsWe have standardized the isolation procedure to obtain a cell population with hepatogenic properties prior to in vivo transplantation. When subjected to hepatogenic differentiation on untreated plastic support, UCMSCs differentiated in hepatocyte-like cells as demonstrated by their morphology, progressive up-regulation of mature hepatocyte markers, glycogen storage, albumin and urea secretion. However, cells seeded on 3D-supports showed a minor or negligible differentiation capacity.UCMSCs-transplanted mice showed a more rapid damage resolution, as shown by histological analysis, with a lower inflammation level and an increased catalase activity compared to CCl4-treated mice.ConclusionsOur findings show that UCMSCs can be reliably isolated, have hepatogenic properties and following systemic administration are able to accelerate the resolution of an acute liver injury without any differentiation and manipulation. These features make UCMSCs strong candidates for future application in regenerative medicine for human acute liver disease.

Validation of the BCLC Prognostic System in Surgical Hepatocellular Cancer Patients

BACKGROUND/AIM Prognosis assessment in surgical patients with hepatocellular carcinoma (HCC) remains controversial. The most widely used HCC prognostic tool is the Barcelona Clinic Liver Cancer (BCLC) classification, but its prognostic ability in surgical patients has not been yet validated. The aim of this study was to investigate the value of known prognostic systems in 400 Italian HCC patients treated with radical surgical therapies. METHODS We analyzed a prospective database collection (400 surgical, 315 nonsurgical patients) observed at a single institution from 2000 and 2007. By using survival times as the only outcome measure (Kaplan-Meier method and Cox regression), the performance of the BCLC classification was compared with that of Okuda, Cancer of the Liver Italian Program, United Network for Organ sharing TNM, and Japan Integrated Staging Score staging systems. RESULTS Two hundred twenty-five patients underwent laparotomy resection; 55, laparoscopic procedures (ablation and/or resection); and 120, liver transplantations. In the surgical group, BCLC proved the best HCC prognostic system. Three-year survival rates of patients in BCLC Stages A, B, and C were 81%, 56%, and 44% respectively, (P < .01); whereas all other tested staging systems did not show significant stratification ability. When all 715 HCC patients were considered, surgery proved to be a significant survival predictor in each BCLC stage (A, B, and C). CONCLUSIONS BCLC staging showed the best interpretation of the survival distribution in a surgical HCC population. The BCLC treatment algorithm should consider the role of surgery also for intermediate-advanced stages of liver disease.

Correlation between the liver temperature employed during machine perfusion and reperfusion damage: Role of Ca2+

This study compares the effects of machine perfusion (MP) at different temperatures with simple cold storage. In addition, the role of Ca2+ levels in the MP medium was evaluated. For MP, rat livers were perfused for 6 hours with Krebs‐Henseleit (KH) solution (with 1.25 or 2.5 mM CaCl2) at 4°C, 10°C, 20°C, 25°C, 30°C, or 37°C. For cold storage, livers were perfused in situ and preserved with Celsior solution at 4°C for 6 hours. The reperfusion period (2 hours at 37°C) was performed under the same conditions used for MP‐preserved and cold storage–preserved livers. Hepatic enzyme release, bile production, adenosine triphosphate (ATP) levels, and morphology were evaluated during MP and reperfusion. MP at 37°C caused marked enzyme release; the same findings were obtained during reperfusion. By contrast, MP temperature lowering induced a significant decrease in liver damage. High levels of biliary gamma‐glutamyltransferase and lactate dehydrogenase were found with MP at 4°C and 10°C but not with MP at 20°C. When a KH–1.25 mM CaCl2 solution was used during MP at 20°C, very low enzyme release was observed and significantly lower hepatic damage was present at the end of the reperfusion period in comparison with cold storage. The same results were obtained when ruthenium red, a calcium uniporter blocker, was added to KH–2.5 mM CaCl2. ATP levels were higher and morphology was better in liver preserved with KH–1.25 mM CaCl2. MP at 20°C with KH–1.25 mM CaCl2 resulted in better quality liver preservation, improving hepatocyte and endothelial biliary cell survival, in comparison with cold storage. This raises the need to reconsider the temperature and calcium levels to be used during liver MP. Liver Transpl 14:494–503, 2008.

Use of N‐acetylcysteine during liver procurement: A prospective randomized controlled study

Antioxidant agents have the potential to reduce ischemia/reperfusion damage to organs for liver transplantation (LT). In this prospective, randomized study, we tested the impact of an infusion of N‐acetylcysteine (NAC) during liver procurement on post‐LT outcomes. Between December 2006 and July 2009, 140 grafts were transplanted into adult candidates with chronic liver disease who were listed for first LT, and according to a sequential, closed‐envelope, single‐blinded procedure, these patients were randomly assigned in a 1/1 ratio to an NAC protocol (69 patients) or to the standard protocol without NAC [71 patients (the control group)]. The NAC protocol included a systemic NAC infusion (30 mg/kg) 1 hour before the beginning of liver procurement and a locoregional NAC infusion (300 mg through the portal vein) just before cross‐clamping. The primary endpoint was graft survival. The graft survival rates at 3 and 12 months were 93% and 90%, respectively, in the NAC group and 82% and 70%, respectively, in the control group (P = 0.02). An adjusted Cox analysis showed a significant NAC effect on graft survival at both 3 months [hazard ratio = 1.65, 95% confidence interval (CI) = 1.01‐2.93, P = 0.04] and 12 months (hazard ratio = 1.73, 95% CI = 1.14‐2.76, P ≤ 0.01). The incidence of postoperative complications was lower in the NAC group (23%) versus the control group (51%, P < 0.01). In the subgroup of 61 patients (44%) receiving suboptimal grafts (donor risk index > 1.8), the incidence of primary dysfunction of the liver was lower (P = 0.09) for the NAC group (15%) versus the control group (32%). In conclusion, the NAC harvesting protocol significantly improves graft survival. The effect of NAC on early graft function and survival seems higher when suboptimal grafts are used. Liver Transpl 19:135–144, 2013.

Subnormothermic Machine Perfusion for Non–Heart-Beating Donor Liver Grafts Preservation in a Swine Model: A New Strategy to Increase the Donor Pool?

We previously reported that subnormothermic machine perfusion (sMP; 20°C) is able to improve the preservation of livers obtained from non-heart-beating donors (NHBDs) in rats. We have compared sMP and standard cold storage (CS) to preserve pig livers after 60 minutes of cardiac arrest. In the sMP group livers were perfused for 6 hours with Celsior at 20°C. In the CS group they were stored in Celsior at 4°C for 6 hours as usual. To simulate liver transplantation, both sMP- and CS-preserved livers were reperfused using a mechanical continuous perfusion system with autologus blood for 2 hours at 37°C. At 120 min after reperfusion aspartate aminotransferase levels in sMP versus CS were 499 ± 198 versus 7648 ± 2806 U/L (P < .01); lactate dehydrogenase 1685 ± 418 versus 12998 ± 3039 U/L (P < .01); and lactic acid 4.78 ± 3.02 versus 10.46 ± 1.79 mmol/L (P < .01) respectively. The sMP group showed better histopathologic results with significantly less hepatic damage. This study confirmed that sMP was able to resuscitate liver grafts from large NHBD animals.

Nanotechnology to drive stem cell commitment

Stem cells (SCs) are undifferentiated cells responsible for the growth, homeostasis and repair of many tissues. The maintenance and survival of SCs is strongly influenced by several stimuli from the local microenvironment. The majority of signaling molecules interact with SCs at the nanoscale level. Therefore, scaffolds with surface nanostructures have potential applications for SCs and in the field of regenerative medicine. Although some strategies have already reached the field of cell biology, strategies based on modification at nanoscale level are new players in the fields of SCs and tissue regeneration. The introduction of the possibility to perform such modifications to these fields is probably due to increasing improvements in nanomaterials for biomedical applications, as well as new insights into SC biology. The aim of the present review is to exhibit the most recent applications of nanostructured materials that drive the commitment of adult SCs for potential clinical applications.

Liver transplantation using suboptimal grafts: Impact of donor harvesting technique

In recent years, an increasing number of suboptimal grafts has been used to reduce the gap between the supply and demand of organs for liver transplantation (LT). In this randomized prospective study, we tested the impact of donor harvesting technique on the posttransplantation outcome of suboptimal donor livers. A modified double perfusion (MDP) technique (aortic and portal cooling with tourniquet clamping of splenomesenteric vein inflow) was compared with the single aortic perfusion (SAP) technique. Between February and November 2005, 35 suboptimal grafts were randomly assigned to either technique (18 MDP livers and 17 SAP livers). Donor and recipient variables were comparable in the 2 study groups. The SAP group had significantly higher blood transaminases and bilirubin levels after LT. The prevalence of graft primary dysfunction (PDF) was also significantly higher (P = 0.01) in the SAP group (35%) than in the MDP group (5%). In 5 cases, all in the SAP group (P = 0.02), early re‐LT (<30 days) was needed. The 6‐month patient and graft survival rates were significantly higher in the MDP (100% in both cases) than in the SAP group (68% and 58%, respectively). The study was stopped in November 2005, when the interim analysis revealed such markedly significant differences between the two groups. In conclusion, the present study showed a very low prevalence of PDF, death, and re‐LT after transplantation with suboptimal liver when a MDP technique was used to harvest the donor graft. Liver Transpl 13:1444–1450, 2007.

Lyn-mediated mitochondrial tyrosine phosphorylation is required to preserve mitochondrial integrity in early liver regeneration.

Functional alterations in mitochondria such as overproduction of ROS (reactive oxygen species) and overloading of calcium, with subsequent change in the membrane potential, are traditionally regarded as pro-apoptotic conditions. Although such events occur in the early phases of LR (liver regeneration) after two-thirds PH (partial hepatectomy), hepatocytes do not undergo apoptosis but continue to proliferate until the mass of the liver is restored. The aim of the present study was to establish whether tyrosine phosphorylation, an emerging mechanism of regulation of mitochondrial function, participates in the response to liver injury following PH and is involved in contrasting mitochondrial pro-apoptotic signalling. Mitochondrial tyrosine phosphorylation, negligible in the quiescent liver, was detected in the early phases of LR with a trend similar to the events heralding mitochondrial apoptosis and was attributed to the tyrosine kinase Lyn, a member of the Src family. Lyn was shown to accumulate in an active form in the mitochondrial intermembrane space, where it was found to be associated with a multiprotein complex. Our results highlight a role for tyrosine phosphorylation in accompanying, and ultimately counteracting, mitochondrial events otherwise leading to apoptosis, hence conveying information required to preserve the mitochondrial integrity during LR.

Prospective Validation of a New Priority Allocation Model for Liver Transplant Candidates: An Interim Analysis

BACKGROUND The system that controls the waiting list (WL) and organ allocation for liver transplantation (OLT) seeks to achieve 3 main goals: objectivity, low dropout risks and good post-OLT results. We sought to prospectively validate a priority allocation model that is believed to achieve objectivity without penalizing dropout risk and post-OLT results. METHODS We evaluated a study group of 272 patients enrolled in 2006-2007. WL candidates were divided into 2 categories: cirrhotic patients classified according to Model for End-Stage Liver Disease (MELD) score (MELD list and patients with hepatocellular carcinoma (HCC) organized according to a specific score (non-MELD list). The allocation algorithm for donor-recipient match assigned an optimal graft to the first MELD candidate with a MELD score of >or=20; a suboptimal graft, to the first non-MELD patient. A respective control group of 327 patients transplanted from 2003-2006 was characterized by a unique WL with a free allocation policy. We performed an interim analysis of this prospectively controlled study. RESULTS Although the study group showed a lower percentage of OLT (P < .05) than the control group (37% vs 45%), it selected patients for OLT based on a higher MELD score (P < .05), thus obtaining similar dropout, post-OLT survivals, and intention-to-treat (ITT) survival probabilities as the controls. Among MELD patients, we observed a significantly reduced dropout and better ITT survival profiles than those of the control group (P = .02), whereas the similar results were delivered among non-MELD patients (P > .05). Among patients with a MELD score of >or=20, the prevalences of suboptimal grafts (0% vs 48%) and of early graft losses (0% vs 21%) were lower in the study than in the control group (P < .05). CONCLUSIONS We prospectively validated a priority allocation model based on objective criteria that achieved high ITT survival rates.