Innovations in 3D bioprinting and biomaterials for liver tissue engineering:Paving the way for tissue-engineered liver

The liver is a pivotal organ that maintains internal homeostasis and actively participates in multiple physiological processes.Liver tissue engineering(LTE),by which in vitro biomimetic liver models are constructed,serves as a platform for disease research,drug screening,and cell replacement therapies.3D bioprinting is used in tissue engineering to create microenvironments that closely mimic authentic tissues with carefully selected functional biomaterials.Ideal functional biomaterials exhibit characteristics such as high biocompatibility,mechanical strength,flexibility,processability,and tunable degradability.Biomaterials can be categorized into natural and synthetic biomaterials,each with its own advantages and limitations,and their combinations serve as a primary source of 3D bioprinting materials.It is noteworthy that the liver decellularized extracellular matrix(dECM),obtained by removing cellular components from tissues,possesses traits such as bioactivity,biocompatibility,and non-immunogenicity,making it a common choice among functional biomaterials.Furthermore,crosslinking of biomaterials significantly impacts the mechanical strength,physicochemical properties,and cellular behavior of the printed structures.This review covers the current utilization of biomaterials in LTE,focusing on natural and synthetic biomaterials as well as the selection and application of crosslinking methods.The aim is to enhance the fidelity of in vitro liver tissue models by providing a comprehensive coverage of functional biomaterials,thereby establishing a versatile platform for tissue-engineered livers.

Liver regeneration using decellularized splenic scaffold: a novel approach in tissue engineering

BACKGROUND： The potential application of decellularized liver scaffold for liver regeneration is limited by severe shortage of donor organs. Attempt of using heterograft scaffold is accompanied with high risks of zoonosis and immunological rejection. We proposed that the spleen, which procured more extensively than the liver, could be an ideal source of decellularized scaffold for liver regeneration. METHODS： After harvested from donor rat, the spleen was processed by 12-hour freezing/thawing ×2 cycles, then circulation perfusion of 0.02% trypsin and 3% Triton X-100 sequentially through the splenic artery for 32 hours in total to prepare decellularized scaffold. The structure and component characteristics of the scaffold were determined by hematoxylin and eosin and immumohistochemical staining, scanning electron microscope, DNA detection, porosity measurement, biocompatibility and cytocompatibility test. Recellularization of scaffold by 5×106 bone marrow mesenchymal stem cells（BMSCs） was carried out to preliminarily evaluate the feasibility of liver regeneration by BMSCs reseeding and differentiation in decellularized splenic scaffold.RESULTS： After decellularization, a translucent scaffold, which retained the gross shape of the spleen, was generated. Histological evaluation and residual DNA quantitation revealed the remaining of extracellular matrix without nucleus and cytoplasm residue. Immunohistochemical study proved the existence of collagens I, IV, fibronectin, laminin and elastin in decellularized splenic scaffold, which showed a similarity with decellularized liver. A scanning electron microscope presented the remaining three-dimensional porous structure of extracellular matrix and small blood vessels. The poros-ity of scaffold, aperture of 45.36±4.87 μm and pore rate of 80.14%±2.99% was suitable for cell engraftment. Subcutaneous implantation of decellularized scaffold presented good histocompatibility, and recellularization of the splenic scaffold demonstrated that BMSCs could locate and survive in the decellularized matrix. CONCLUSION： Considering the more extensive organ source and satisfying biocompatibility, the present study indicated that the three-dimensional decellularized splenic scaffold might have considerable potential for liver regeneration when combined with BMSCs reseeding and differentiation.

Green Electrospun Silk Fibroin/Galactose Chitosan Composite Nanofibrous Scaffolds for Hepatic Tissue Engineering

The electrospun nanofibrous scaffolds made of proteins and polysaccharides were thought to be able to simulate the structure of natural extracellular matrix well.Silk fibroin(SF)and chitosan(CS)are probably the most widely used natural materials in biomedical fields including liver tissue engineering for their good properties and wide variety of sources.The asialoglycoprotein receptors of hepatocyte were reported to specifically recognize and interact with galactose.In this work,a green electrospun SF/galactosylated chitosan(GC)composite nanofibrous scaffold was fabricated and characterized.The data indicated that the addition of GC greatly influenced the spinning effect of SF aqueous solution,and the average diameter of the composite nanofibers was about 520nm.Moreover,the green electrospun SF/GC nanofibrous scaffolds were demonstrated significantly enhancing the adhesion and proliferation of hepatocyte(RH35)according to our data.The present study did a useful exploration on constructing scaffolds for liver regeneration by green electrospinning,and also laid a good foundation for the further applicative research of this green electrospun scaffolds in liver tissue engineering.

Noncoding RNAs and Their Potential Therapeutic Applications in Tissue Engineering

Tissue engineering is a relatively new but rapidly developing field in the medical sciences. Noncoding RNAs(ncRNAs) are functional RNA molecules without a protein-coding function; they can regulate cellular behavior and change the biological milieu of the tissue. The application of ncRNAs in tissue engineering is starting to attract increasing attention as a means of resolving a large number of unmet healthcare needs, although ncRNA-based approaches have not yet entered clinical practice. In-depth research on the regulation and delivery of ncRNAs may improve their application in tissue engineering.The aim of this review is: to outline essential ncRNAs that are related to tissue engineering for the repair and regeneration of nerve, skin, liver, vascular system, and muscle tissue; to discuss their regulation and delivery; and to anticipate their potential therapeutic applications.

Polyethylene glycol crosslinked decellularized single liver lobe scaffolds with vascular endothelial growth factor promotes angiogenesis in vivo

Background: Improving the mechanical properties and angiogenesis of acellular scaffolds before transplantation is an important challenge facing the development of acellular liver grafts. The present study aimed to evaluate the cytotoxicity and angiogenesis of polyethylene glycol(PEG) crosslinked decellularized single liver lobe scaffolds(DLSs), and establish its suitability as a graft for long-term liver tissue engineering. Methods: Using mercaptoacrylate produced by the Michael addition reaction, DLSs were first modified using N-succinimidyl S-acetylthioacetate(SATA), followed by cross-linking with PEG as well as vascular endothelial growth factor(VEGF). The optimal concentration of agents and time of the individual steps were identified in this procedure through biomechanical testing and morphological analysis. Subsequently, human umbilical vein endothelial cells(HUVECs) were seeded on the PEG crosslinked scaffolds to detect the proliferation and viability of cells. The scaffolds were then transplanted into the subcutaneous tissue of Sprague-Dawley rats to evaluate angiogenesis. In addition, the average number of blood vessels was evaluated in the grafts with or without PEG at days 7, 14, and 21 after implantation. Results: The PEG crosslinked DLS maintained their three-dimensional structure and were more translucent after decellularization than native DLS, which presented a denser and more porous network structure. The results for Young’s modulus proved that the mechanical properties of 0.5 PEG crosslinked DLS were the best and close to that of native livers. The PEG-VEGF-DLS could better promote cell proliferation and differentiation of HUVECs compared with the groups without PEG cross-linking. Importantly, the average density of blood vessels was higher in the PEG-VEGF-DLS than that in other groups at days 7, 14, and 21 after implantation in vivo. Conclusions: The PEG crosslinked DLS with VEGF could improve the biomechanical properties of native DLS, and most importantly, their lack of cytotoxicity provides a new route to promote the proliferation of cells in vitro and angiogenesis in vivo in liver tissue engineering.

Advances in liver engineering with cell, scaffold, and vascularization

Liver tissue engineering is a promising alternative to organ transplantation for the treatment of end-stage liver disease,acute liver failure,and liver-based metabolic disorders.This review discusses the current progress and obstacles in tissue engineering of cell sources,biomaterials,and vascularization strategies,which are key to constructing functional liver tissues.Significant progress has been made in developing available cell sources,tunable biomaterials,and integrating vascular networks.However,challenges remain in replicating the complex architecture and function of the liver.Further research is required to overcome these hurdles and ultimately realize the full potential of tissue-engineered liver constructs in clinical applications.

Long-term functional maintenance of primary hepatocytes in vitro using macroporous hydrogels engineered through liquid-liquid phase separation

Preserving the functionality of hepatocytes in vitro poses a significant challenge in liver tissue engineering and bioartificial liver,as these cells rapidly lose their metabolic and functional characteristics after isolation.Inspired by the macroporous structures found in native liver tissues,here we develop synthetic hydrogel scaffolds that closely mimic the liver’s structural organization through the phase separation between polyethylene glycol(PEG)and polysaccharides.Our hydrogels exhibit interconnected macroporous structures and appropriate mechanical properties,providing an optimal microenvironment conducive to hepatocyte adhesion and the formation of sizable aggregates.Compared to two-dimensional hepatocyte cultures,enhanced functionalities of hepatocytes cultured in our macroporous hydrogels were observed for 14 days,as evidenced by quantitative reverse-transcription–polymerase chain reactions(qRT-PCR),immunofluorescence,and enzyme linked immunosorbent assay(ELISA)analyses.Protein sequencing data further confirmed the establishment of cell–cell interactions among hepatocytes when cultured in our hydrogels.Notably,these hepatocytes maintained a protein expression lineage that closely resembled freshly isolated hepatocytes,particularly in the Notch and tumor necrosis factor(TNF)signaling pathways.These results suggest that the macroporous hydrogels are attractive scaffolds for liver tissue engineering.

Biomaterial Scaffolds with Biomimetic Fluidic Channels for Hepatocyte Culture

Biomaterial scaffolds play an important role in maintaining the viability and biological functions of highly metabolic hepatocytes in liver tissue engineering. One of the major challenges involves building a complex microchannel network inside three-dimensional （3D） scaffolds for efficient mass transportation. Here we presented a biomimetic strategy to generate a mi- crochannel network within porous biomaterial scaffolds by mimicking the vascular tree of rat liver. The typical parameters of the blood vessels were incorporated into the biomimetic design of the microchannel network such as branching angle and diameter. Silk fibroin-gelatin scaffolds with biomimetic vascular tree were fabricated by combining micromolding, freeze drying and 3D rolling techniques. The relationship between the micro-channeled design and flow pattern was revealed by a flow experiment, which indicated that the scaffolds with biomimetic vascular tree exhibited unique capability in improving mass transportation inside the 3D scaffold. The 3D scaffolds, preseeded with primary hepatocytes, were dynamically cultured in a bioreactor system. The results confirmed that the pre-designed biomimetic microchannel network facilitated the generation and expansion of hepatocytes.