Functional Recellularization of Acellular Rat Liver Scaffold by Induced Pluripotent Stem Cells: Molecular Evidence for Wnt/B-Catenin Upregulation

Background. Liver transplantation remains the only viable therapy for liver failure but has a severely restricted utility. Here, we aimed to decellularize rat livers to form acellular 3D bio-scaffolds suitable for seeding with induced pluripotent cells (iPSCs) as a tool to investigate the role of Wnt/β-catenin signaling in liver development and generation. Methods. Dissected rat livers were randomly divided into three groups: I (control); II (decellularized scaffolds) and III (recellularized scaffolds). Liver decellularization was established via an adapted perfusion procedure and assessed through the measurement of extracellular matrix (ECM) proteins and DNA content. Liver recellularization was assessed through histological examination and measurement of transcript levels of Wnt/β-catenin pathway, hepatogenesis, liver-specific microRNAs and growth factors essential for liver development. Adult rat liver decellularization was confirmed by the maintenance of ECM proteins and persistence of growth factors essential for liver regeneration. Results. iPSCs seeded rat decellularized livers displayed upregulated transcript expression of Wnt/β-catenin pathway-related, growth factors, and liver specification genes. Further, recellularized livers displayed restored liver-specific functions including albumin secretion and urea synthesis. Conclusion. This establishes proof-of-principle for the generation of three-dimensional liver organ scaffolds as grafts and functional re-establishment.

Functional characterization of a bioengineered liver after heterotopic implantation in pigs

Organ bioengineering offers a promising solution to the persistent shortage of donor organs. However, the progression of this technology toward clinical use has been hindered by the challenges of reconstituting a functional vascular network, directing the engraftment of specific functional cell types, and defining appropriate culture conditions to concurrently support the health and phenotypic stability of diverse cell lineages. We previously demonstrated the ability to functionally reendothelialize the vasculature of a clinically scaled decellularized liver scaffold with human umbilical vein endothelial cells (HUVECs) and to sustain continuous perfusion in a large animal recovery model. We now report a method for seeding and engrafting primary porcine hepatocytes into a bioengineered liver (BEL) scaffold previously reendothelialized with HUVECs. The resulting BELs were competent for albumin production, ammonia detoxification and urea synthesis, indicating the presence of a functional hepatocyte compartment. BELs additionally slowed ammonia accumulation during in vivo perfusion in a porcine model of surgically induced acute liver failure. Following explant of the graft, BEL parenchyma showed maintenance of canonical endothelial and hepatocyte markers. Taken together, these results support the feasibility of engineering a clinically scaled functional BEL and establish a platform for optimizing the seeding and engraftment of additional liver specific cells.

AuCu nanofibers for electrosynthesis of urea from carbon dioxide and nitrite

Carbon dioxide reduction reaction (CO2RR) is a promising technology for mitigating greenhouse gas emission and achieving carbon neutrality. However, coupling CO2RR with other reactions to produce high value-added chemicals remains a challenge. In this work, we report self-assembled nanofibers composed of ultra-thin AuCu alloy nanowires possessing a Boerdijk-Coxeter structure with (111)-dominant facets for the electrosynthesis of urea by coupling CO2RR with nitrite reduction reaction (NO2−RR). The rich structural defects and AuCu bimetallic alloy composition provide a large number of highly catalytically active sites. The constructed AuCu nanofibers display excellent urea synthesis performance in the electrolyte solution containing 0.01 M KNO2 with continuous drumming of CO2, achieving a high urea yield rate of up to 3889.6 µg h− 1 mg− 1cat. and a high Faraday efficiency of 24.7% at -0.9 V. This work provides a feasible method for the rational design of self-assembled bimetallic nanofibers for electrosynthesis of urea under ambient conditions.

INCREASING THE EFFICIENCY OF THE SYSTEMS OF COMPRESSOR-PUMPING AND REFRIGERATION UNITS SUPPLYING LIQUID CO2 AND NH3 TO THE UNIT FOR CARBAMIDE SYNTHESIS

Carbon dioxide is used in large volumes to produce urea, a highly efficient nitrogen fertilizer. It is compressed in a multistage compressor to a pressure of 15 MPa and fed to the urea synthesis unit. The specific energy consumption for the compression of carbon dioxide by a compressor reaches 0.16 kWh/kg. It may be more profitable to use in the system of compressor-pumping and refrigeration units. They can be used to liquefy carbon dioxide and compress it to pressure 15 MPa before feeding it to the synthesis of urea. In the simplest scheme, an ammonia compression refrigeration machine (ACRM) is included in the system to improve efficiency. The specific energy consumption in such a system for the liquefaction and compression of CO2 is 0.118 kWh/kg. In case of replacement of the ACRM with an absorption refrigeration machine, unit costs can be reduced to 0.09 kWh/kg. These two systems can be used to increase urea production or to ensure stable operation of the units during the summer period of their operation. The analysis showed that further improvement of the technological scheme of the entire system will completely abandon the use of the compressor method of compression of CO2 to pressure 15 MPa before its supply to the urea synthesis unit. To do this, you need to include an additional absorption lithium bromide refrigeration machine in the system. In this scheme, the compressor-pumping unit will provide the simultaneous supply of liquid carbon dioxide and ammonia for the synthesis of urea with a pressure of 15 MPa. To increase the daily production of urea from 1400 to 2000 tons, it is necessary to increase the feed liquid CO2 in the amount of 62 t/hour and liquid NH3 — 47.5 t/hour. Bibl. 14, Fig. 3.

Role of the cardiovascular system in ammonia excretion in early life stages of zebrafish (Danio rerio)

The purpose of this study was to investigate if the cardiovascular system is important for ammonia excretion in the early life stages of zebrafish. Morpholino knockdowns of cardiac troponin T (TNNT2) or vascular endothelial growth factor (VEGFA) provided morphants with non-functional circulation. At the embryonic stage (30-36 hpf), ammonia excretion was not constrained by lack of cardiovascular function. At 2 days post fertilization (dpf) and 4 dpf, morpholino knockdowns of TNNT2 or VEGFA significantly reduced ammonia excretion in all morphants. Expression of rhag, rhbg and rhcgb showed no significant changes but the mRNA levels of the urea transporter (ut) were upregulated in the 4 dpf morphants. Taken together, rhag, rhbg, rhcgb and ut gene expression and an unchanged tissue ammonia concentration but an increased tissue urea concentration, suggest that impaired ammonia excretion led to increased urea synthesis. However, in larvae anesthetized with tricaine or clove oil, ammonia excretion was not reduced in the 4 dpf morphants compared to controls. Further, oxygen consumption was reduced in morphants regardless of anesthesia. These results suggest that cardiovascular function is not directly involved in ammonia excretion, but rather reduced activity and external convection may explain reduced ammonia excretion and compensatory urea accumulation in morphants with reduced cardiovascular function.

Electrospun Microfibers Modulate Intracellular Amino Acids in Liver Cells via Integrin β1

Although numerous recent studies have shown the importance of polymeric microfibrous extracellular matrices (ECMs) in maintaining cell behaviors and functions, the mechanistic nexus between ECMs and intracellular activities is largely unknown. Nevertheless, this knowledge will be critical in understanding and treating diseases with ECM remodeling. Therefore, we present our findings that ECM microstructures could regulate intracellular amino acid levels in liver cells mechanistically through integrin β1. Amino acids were studied because they are the fundamental blocks for protein synthesis and metabolism, two vital functions of liver cells. Two ECM conditions, flat and microfibrous, were prepared and studied. In addition to characterizing cell growth, albumin production, urea synthesis, and cytochrome p450 activity, we found that the microfibrous ECM generally upregulated the intracellular amino acid levels. Further explorations showed that cells on the flat substrate expressed more integrin β1 than cells on the microfibers. Moreover, after partially blocking integrin β1 in cells on the flat substrate, the intracellular amino acid levels were restored, strongly supporting integrin β1 as the linking mechanism. This is the first study to report that a non-biological polymer matrix could regulate intracellular amino acid patterns through integrin. The results will help with future therapy development for liver diseases with ECM changes (e.g., fibrosis).