Decellularized liver-regenerative 3D printing biomaterials for cell-based liver therapies via a designed procedure combined with papain-containing reagent treatments and supercritical fluids

BACKGROUND: The biologic scaffolds derived from decellularized tissues and organs have been successfully developed in a variety of preclinical and/or clinical studies. OBJECTIVE: The new decellularized liver-regenerative 3D printing biomaterials were designed and prepared for cell-based liver therapies. METHODS: An extraction process was employed to remove the tissue and cellular molecules from porcine liver via pretreatment of supercritical fluid of carbon dioxide (ScCO2). Varying porosities of the decellularized liver tissues were created using papain-containing reagent treatments after ScCO2. RESULTS: The resulting liver-regenerative 3D printing biomaterials of decellularized liver collagen scaffolds were characterized by Fourier transform infrared spectroscopy, thermo-gravimetric analysis, differential scanning calorimetry and scanning electron microscopy. CONCLUSIONS: The decellularized liver collagen scaffolds with good thermal stability (>150 °C) were obtained and employed as liver-regenerative 3D printing biomaterials for cell-based liver therapies.

Portal hypertension is the main driver of liver stiffness in advanced liver cirrhosis

Liver stiffness (LS) is a novel non-invasive parameter widely used in clinical hepatology. LS correlates with liver fibrosis stage in non-cirrhotic patients. In cirrhotic patients it also shows good correlation with Hepatic Venous Pressure Gradient (HVPG). Our aim was to assess the contribution of liver fibrosis and portal hypertension to LS in patients with advanced liver cirrhosis. Eighty-one liver transplant candidates with liver cirrhosis of various aetiologies underwent direct HVPG and LS measurement by 2D shear-wave elastography (Aixplorer Multiwave, Supersonic Imagine, France). Liver collagen content was assessed in the explanted liver as collagen proportionate area (CPA) and hydroxyproline content (HP). The studied cohort included predominantly patients with Child-Pugh class B and C (63/81, 77.8 %), minority of patients were Child-Pugh A (18/81, 22.2 %). LS showed the best correlation with HVPG (r=0.719, p<0.001), correlation of LS with CPA (r=0.441, p<0.001) and HP/Amino Acids (r=0.414, p< 0.001) was weaker. Both variables expressing liver collagen content showed good correlation with each other (r=0.574, p<0.001). Multiple linear regression identified the strongest association between LS and HVPG (p<0.0001) and weaker association of LS with CPA (p = 0.01883). Stepwise modelling showed minimal increase in r2 after addition of CPA to HVPG (0.5073 vs. 0.5513). The derived formula expressing LS value formation is: LS=2.48 + (1.29 x HVPG) + (0.26 x CPA). We conclude that LS is determined predominantly by HVPG in patients with advanced liver cirrhosis whereas contribution of liver collagen content is relatively low.

Isoniazid and Rifampicin Produce Hepatic Fibrosis through an Oxidative Stress-Dependent Mechanism

Background & Aims. Chronic hepatitis (CH) has emerged as a distinct outcome of drug-induced liver injury (DILI). Combination therapy of Isoniazid (INH) and Rifampicin (RMP) which is widely used for prolonged periods can cause acute hepatotoxicity and has been also incriminated in chronic DILI. We sought evidence of the production of hepatic fibrosis on long-term INH-RMP treatment through experiments in BALB/c mice exposed to INH-RMP. Methods. A combined dose of INH (50 mg) and RMP (100 mg) per kg body weight per day was administered to mice by oral gavage, 6 days a week, for 4 to 24 weeks for the assessment of liver injury, oxidative stress, and development of hepatic fibrosis, including demonstration of changes in key fibrogenesis linked pathways and mediators. Results. Progressive increase in markers of hepatic stellate cell (HSC) activation associated with changes in matrix turnover was observed between 12 and 24 weeks of INH-RMP treatment along with the elevation of liver collagen content and significant periportal fibrosis. These were associated with concurrent apoptosis of the hepatocytes, increase in hepatic cytochrome P450 2E1 (CYP2E1), NADPH oxidase (NOX) activity, and development of hepatic oxidative stress. Conclusions. INH-RMP can activate HSC through generation of NOX-mediated oxidative stress, leading to the development of liver fibrosis.

Interesterified Fats Induce Deleterious Effects on Adipose Tissue and Liver in LDLr-KO Mice

Interesterified fats are being widely used by the food industry in an attempt to replace trans fatty acids. The effect of interesterified fats containing palmitic or stearic acids on lipid metabolism and inflammatory signaling pathways in adipose and hepatic tissues was evaluated. Male LDLr-KO mice were fed a high-fat diet containing polyunsaturated (PUFA), palmitic (PALM), palmitic interesterified (PALM INTER), stearic (STEAR), or stearic interesterified (STEAR INTER) fats for 16 weeks. The expression of genes and protein levels involved in lipid metabolism and inflammatory processes in liver and white adipose tissue was determined by quantitative RT-PCR and by Western blot, respectively. The infiltration of inflammatory cells in hepatic and adipose tissues was determined by eosin and hematoxylin, while liver collagen content was determined by Sirius Red staining. Both interesterified fats increased liver collagen content and JNK phosphorylation. Additionally, the STEAR INTER group developed nonalcoholic steatohepatitis (NASH) associated with higher neutrophil infiltration. PALM INTER induced adipose tissue expansion and enlargement of adipocytes. Furthermore, PALM INTER triggered increased IKK phosphorylation and TNFα protein content, conditions associated with the upstream activation of the NFkB signaling pathway. STEAR INTER induced NASH, while PALM INTER triggered hepatic fibrosis and adipocyte hypertrophy with inflammatory response in LDLr-KO mice.

Tracer-based estimates of protein flux in cases of incomplete product renewal: evidence and implications of heterogeneity in collagen turnover

The synthesis of various molecules can be estimated by measuring the incorporation of a labeled precursor into a product of interest. Unfortunately, a central problem in many studies has been an inability to estimate the intracellular dilution of the precursor and therein correctly calculate the synthesis of the product; it is generally assumed that measuring the true product labeling is straightforward. We initiated a study to examine liver collagen synthesis and identified an apparent problem with assumptions regarding measurements of the product labeling. Since it is well known that collagen production is relatively slow, we relied on the use of [2H]H2O labeling (analogous to a primed infusion) and sampled animals over the course of 16 days. Although the water labeling (the precursor) remained stable and we observed the incorporation of labeled amino acids into collagen, the asymptotic protein labeling was considerably lower than what would be expected based on the precursor labeling. Although this observation is not necessarily surprising (i.e., one might expect that a substantial fraction of the collagen pool would appear “inert” or turn over at a very slow rate), its implications are of interest in certain areas. Herein, we discuss a novel situation in which tracers are used to quantify rates of flux under conditions where a product may not undergo complete replacement. We demonstrate how heterogeneity in the product pool can lead one to the wrong conclusions regarding estimates of flux, and we outline an approach that may help to minimize errors surrounding data interpretation.