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Tissue engineering has followed the 4-component paradigm of biomaterials, cells, in vitro constructs, and integration into living hosts or other applications for the past few decades . Each component is independently researched and the final applications depend on the available components off-the-shelf. The approach is successful for engineering relatively simple tissues whose functional performance does not strictly depend on the detailed structural features. E.g. early work on tissue-engineered skin has led to commercial products in clinical-uses today that cover the wound and prevent infection without concerns for the fine skin-features such as wrinkles or hair follicles that are important for aesthetics and perspiration . Biomaterials research has benefitted from industrial materials research adding biocompatibility and biodegradability. Liver tissue engineering research started with the development of hybrid liver-support systems  and cell-seeded scaffolds for stimulating liver regeneration . The field has since progressed quickly into more sophisticated engineering approaches since liver is a complex organ with vital functions that depend on the structural features at single-cell dimensions. Without fine controls of these structural features such as bile canaliculi, sinusoids, cell shapes and polarity, tissue functions are not restored in predictable ways. The fine controls require precision engineering of the microenvironments in which the cells reside; such microenvironments are synthetic biomaterials, small molecules or neighboring cells. In a historically separate but merged field of regenerative medicine, stem cells and stem cell-derived liver cells have been engineered for implantation or transplantation . Therefore, the boundary between biomaterials and cellular engineering research has blurred recently .
Liver tissue engineering applications currently consolidate into three main concerns: 1) toolbox (biomaterials, cells, and constructs) development for precision liver tissue engineering; 2) in vivo liver regeneration; and 3) in vitro testing of xenobiotics (e.g. drugs and pathogens). In this review we will discuss the trends in these 3 areas, and an emerging systems-level approach for rational design of biomaterials for applications.
Toolbox development for precision liver tissue engineering
Traditional tissue engineering strategies employ a ''top-down'' approach, in which cells are assembled onto a macroscopic polymeric scaffold with feature size in the range of mm-cm [7, 8]. As the scaffold degrades, the cells and their secreted extracellular matrix (ECM) fill in the void and eventually form tissue-like structures . Top-down approaches often have difficulty recreating the intricate micro-structural features of tissues .
Biomaterials for cellular assembly and micro-scale controls of liver cells (micro-gel & linker)
Liver contains repeating micro-scale structures that are amenable to micro-scale controls and bottom-up approaches (Figure 1). Earlier works have used micro-contact printing to create hepatocyte spheroids . Micro-scale controls of hepatocytes by patterning have improved their functions in extended culture [12, 13]. Micro-engineered micro-gels with sizes between <1 µm (sub-cellular mesoscale) to >1 cm (tissue-scale) [9, 14] were developed. The precise control of mesoscale self-assembly with desired orientation of cell-laden micro-gels was achieved in a scalable manner [15, 16]. Polyethylene glycol (PEG)-based cell encapsulation allowed the construction of micro-structural features within a three-dimensional (3D) cell-hydrogel network. Liver cells suspended in polymer solution were photo-immobilized locally in controlled hydrogel architecture forming functional 3D hepatic construct with complex internal features . Recently, photo-protected moieties bound within hydrogels were exploited to support cellular activities . The hydrogels were also free-form patterned with cell-responsive ligands using confocal microscopy, with reported feature sizes as small as 5µm . Anseth et al generated hydrogels which possessed both chemical and mechanical patterns through degradation of the photocleavable hydrogel to control the migration of stem cells within the hydrogel via temporal and spatial controls over photodegradation .
Self-assembled aggregation  or cell-sheet engineering offers alternative approaches to micro-scale controls . Synthetic linkers serve as 'cell glues' to connect individual cells in a gel-free environment with high consistency to form 3D multi-cellular constructs. Cell surfaces are modified with non-native functional groups as reactive handles for synthetic linkers such as PEI hydrazide  or dendrimer hydrazide. C3A cells are rapidly assembled within a minute into multi-cell structures using a dendrimeric linker with membrane-insertion group . The linker stabilizes the multi-cellular structures of defined shapes and patterns in gel-free environment by mechanically confining the cells. Recently, complementary DNA sequences attached to amphiphilic PEG-lipids are inserted into the membranes of two cell populations. Specific cell-cell attachments are induced via hybridization between the two complementary polyDNAs for analyzing and manipulating homogeneous and heterogeneous cell-cell interactions . Cell sheets of hepatocytes are assembled into larger tissue constructs through a number of methods such as random packing, stacking of layers, or directed assembly. One such method involves co-culture of hepatocytes and endothelial cells on patterned dual thermal-responsive polymers with enhanced survival and functionality influenced by the cell-cell interactions . Bottom-up approaches aim to engineer individual liver acini and multiplex to create larger constructs .
Engineering cells for liver tissue construction
Hepatocytes (mature or progenitor, cryopreserved or fresh) and stem cells (adult, fetal, embryonic or iPS) have been extensively investigated [26, 27] as potential cell sources for liver tissue engineering. Hepatocytes exhibit a spectrum of functions but rapidly lose them in culture. Embryonic stem cells can be expanded efficiently  but differentiation into hepatocytes pause at fetal hepatocyte-like stage lacking high levels of mature phenotypes. The cells express a low CYP3A4/3A7 ratio which represents a more fetal hepatocytes-like population. Spatial control of these cells by culturing them as spheroids in defined differentiation media has slightly improved CYP3A4/3A7 ratio . The discovery of iPS cells  balances the ethical, safety and availability issues faced by the other stem cells. Advances are rapid in generating new iPS cells, and differentiating them into hepatocytes ; and to model metabolic diseases of the liver in vitro . Combining the potential of stem cells with appropriate scaffolds with biomaterials such as PLLA/PGA copolymers and Matrigel also facilitated differentiation of ESCs into hepatocyte-like cells (HLCs) with good levels of glycogen production, LDL uptake, rapid increase in Albumin synthesis and increase in certain basal level gene expressions .
Micro-electromechanical systems (MEMS) and nano-technologies
Adopted from semiconductor industry, MEMS technologies enable precision control of spatial distribution of bio-molecules and substrata topography at micro- to nanometer resolution . Micro- or nano-pillars with various features and rigidity have been developed as cell-culture substrata . Multilayer polydimethylsiloxane networks can maintain hepatocyte functions in vitro . Microfluidic 3D cell-culture system (-FCCS) was developed to maintain 3D cell morphology and functions with ECM support  or in gel-free microenvironment . Micro-encapsulated cytokines were control-delivered in microenvironments to locally regulate the cell responses in a PBPK chip with multiple cell types . Ultra-thin microfabricated porous silicon nitride (Si3N4) membranes  immobilized with galactose ligands could enhance hepatocyte functions in sandwich culture. Micro-fabricated devices allow co-culture of hepatocytes with fibroblasts  and endothelial cells  with maximal cell-cell interactions.
Electro-spun nano-fiber mesh (100nm-5µm) with galactosylated poly(Îµ-caprolactone-co-ethyl ethylene phosphate) (PCLEEP) was generated for favorable hepatocyte functions . Nano-pillars or nano-trenches with nanolithography methods such as nanoimprint lithography (NIL) are increasingly employed to support cellular functions . We foresee that nanotechnologies will enable precision engineering of cellular shapes, polarity, spatial connection of bile canaliculi, and other liver tissue structures for functional maintenance. The toolbox of the coming decade would likely migrate from the cellular or multi-cellular resolutions of today's technologies towards sub-cellular mesoscale controls to recreate tissue structures and functions.
In vivo liver regeneration
While the enabling technologies or toolbox become more sophisticated, the integration into useful solutions for final applications has become more purpose-driven (Table 1). There are two principal purposes: 1) functional restoration by delivering engineered cells and tissue constructs in vivo; or 2) stimulating liver's natural regeneration capability by altering the liver's microenvironment with transient but large doses of stimuli. There are corresponding biomaterials and engineering configurations designed for each purpose.
Functional restoration by delivering engineered cells & tissue constructs in vivo
The primary concern here is the engraftment efficiency and survival of the transplanted cells or engineered constructs in hostile disease environment . Repopulating rat livers using reversible immortalized hepatocytes  protect them from imminent death in the hostile environment of the host. Stem cells and liver progenitor cells with possible immune privilege status could enhance engraftment [45, 46]. Injectable fibrin-gel immobilized hepatocyte system facilitates intrahepatic transplantation of hepatocytes and integration in vivo . Advances in scaffolds designed to stimulate angiogenesis and creation of AV-loops in other organs  has been adapted for liver to prolong survival of the transplanted cells. Supplementation of the biomaterial with angiogenic growth factors like VEGF , FGF, PDGF or using pre-vascularized scaffolds have also been exploited . Micro-scale technologies introduced microvasculature into scaffolds for liver tissue engineering . Decellularized liver matrix support hepatocytes ex vivo and maintain high levels of cellular functions in vivo . Co-culture of hepatocytes and non-parenchymal cells on decellularized scaffolds could also establish microvasculature and enhance vascular integrity in the host. A careful and systematic analysis of the ECM composition and distribution of the decellularized liver matrix could lead to novel biomaterials and engineering configurations for stable restoration of liver functions in vivo.
Facilitating liver regeneration by altering liver's microenvironment with transient but large doses of stimuli
Liver's natural regenerative capability is stimulated through strategies such as the delivery of growth factors, drugs and transient genetic modifications [52-54] to modify liver's microenvironment. Long-term gene expression or cell engraftment is less important here. Delivery vehicles were designed to transiently deliver siRNA against a collagen-specific chaperone to hepatic stellate cells to inhibit fibrosis . A single large dose of hepatocytes can also sufficiently shock the microenvironment to regress fibrosis [54, 56]. Highly functional mature hepatocytes with poor engraftment efficiency [26, 57, 58] are more effective in altering the liver's microenvironment than the less functional stem or progenitor cells with high engraftment efficiency. Studies on TGF-¢1 activation pathway demonstrated well-defined thresholds  that decide the course of disease progression or regression/regeneration. Long-term concerns on biomaterials and construct properties (e.g. biodegradability and immune-tolerance) yield to the short-term concerns for effective delivery and mass transport here.
In vitro testing of xenobiotics
Drug development and toxicity testing
In pharmaceutical industry, costs have driven safety testing earlier. Whereas twenty years ago efficacy and potency at the pharmacological target were the sole aim of early work, with ADME and toxicity/pathology determined toward the end of preclinical testing, at present lead compound selection and optimization usually includes assessment of cytotoxicity and increasingly characterization of hepatic ADME and toxicity in vitro. Typically candidate compounds are available only in mg quantities, so in vivo testing is not possible until one has selected a lead. Cryopreservation of human hepatocytes has made testing more convenient, and provided human assays, particularly CYP and transporter inhibition and induction assays, that often behave differently than in preclinical species. When compounds are metabolized differently or inhibit these enzymes or induce/repress these genes differentially in humans, there is the potential for human toxicity and also for predicting drug interactions. Unfortunately, many hepatocyte models were developed with only one or a few endpoints in mind. For example, CYP induction and inhibition assays perform well in monolayer cultures, but lose many other important biochemical pathways . Similarly, sandwich cultures were developed to study canalicular transport, but often lack other enzymes, transporters and CYPs that might be critical in the response to a studied compound. A major issue with many hepatocyte models is their specialization; can they be generalized to detect other compound issues? While a model often is useful with only one purpose, an optimized and general model allowing many, often interacting endpoints, is preferable. In addition to Phase I metabolism (mostly well established CYPs) and transporters , there is increasing interest in Phase II and oxidative stress/ reactive metabolite (OS/RM)-protective enzymes; compounds and their reactive metabolites producing OS/RM generally activate the transcription factor Nrf2 which binds to the antioxidant response element (ARE), which is common to the regulation of many conjugation and OS/RM-handling enzymes. Many idiosyncratic hepatotoxicants produce robust OS/RM, which are well handled by preclinical species and the vast majority of human patients. Inductions of OS/RM-protective enzymes are mostly lost in many hepatocyte models, although covalent binding assays, glutathione conjugation assays, and an Nrf2-reporter assay provide simplified assays for initially screening such compounds. Reactive acyl glucuronides seems to account for other idiosyncratic hepatotoxicity, such as noted for a number of NSAIDs; again simplified chemical assays of reactivity of these compounds is the present screen, but ignores effects that may dominate in hepatocytes. While providing robust physiological ADME properties in a hepatocyte model frequently converts an unreliable cytotoxicity assay (run in cell lines) into a more specific and sensitive human cytotoxicity assay, or for example, provides a more meaningful cholestasis assay, there are other toxicity assays (for example, steatosis and phospholipidosis) that need improvement for modeling human responses. Dykens and Will  have cautioned that using the traditional high glucose culture media eliminates much of the mitochondrial toxicity of compounds due to lack of cellular respiration, so improvements can be made in hepatocyte cultures at fundamental levels. Increasingly cell models have taken the place of animals in safety testing for cosmetics and pharmaceuticals, and in the European Union in particular, there has been a concerted push to develop better in vitro models in response to new laws and statues limiting animal use
In view of these concerns, models have been developed which help better predict different forms of toxicity. Zonation of the liver due to differences in oxygen tension leads to variations in cytochrome expression levels thereby differences in toxicity. Flat plate bioreactors with co-cultured hepatocytes were developed which mimic the differences in cytochrome expression levels, leading to differences in toxicity within different zones in vitro . Better prediction of acute toxicity was demonstrated in micro-scale devices with micro-tissue like constructs in perfusion bioreactors. These devices demonstrated improved hepatocyte functions and sensitivity to cytochrome P450 inducers, and the matching in vitro and in vivo rates of testosterone metabolism . Microfluidic chips have recently been explored to determine the acute toxicity (IC50) and drug clearance of hepatotoxic drugs [36, 65]. Spheroid cultures are garnering increasing attention due to the ability to better maintain cellular functions, but are hindered by their variable drug access characteristics. A pre-spheroid 3D monolayer could address some of these issues . Chronic toxicity is more difficult to study in vitro. Recently developed micro-scale co-cultured hepatocyte device that expresses high levels of functionality for up to 42 days was used to study this form of toxicity . The maintenance of bile canaliculi-like structures in a long-term culture opens up new avenues to study transporters and cytochrome-mediated interactions simultaneously with enhanced accuracy. However these models do not predict idiosyncratic toxicity in vitro. Idiosyncratic toxicity has various underlying causes of which only the inflammation-mediated toxicity can be studied in vitro, though with low predictability . The newly developed in vitro models should be exploited to enable better predictability on phase2 and OS/RM enzymes responses to test a broader range of idiosyncratic toxicants. The trend of testing drugs early would also impose huge requirements for the abundance of high quality cell sources; the robustness, scalability and throughput of the drug testing platforms to be further developed.
Cell models for pathogen testing
Non-availability of efficacious vaccines due to the lack of in vitro models to facilitate Hepatitis C viral replication has drawn increasing attention. Hepatitis C virus can propagate in various hepatoma cell lines but these models do not represent the true characteristics of virus infection. Primary human hepatocytes represent the most physiologically relevant model to study the disease in vitro. The viral entry into cells is controlled by cell polarization and cellular localization of CD-81, CLDN-1 and SCRB-1 [68, 69]. This implies the need for proper structural features recapitulated in spheroids or micro-patterned co-cultures to achieve virus infection and replication in vitro [70, 71]. Research is ongoing to screen novel therapeutics in in vitro cultures and to determine if adaptive viral mutations in vitro are representative of that observed in vivo. Similar models can be developed to test Hepatitis A-E and other liver pathogens such as Plasmodium falciparum. Repopulation of mouse liver with human hepatocytes have shown chimerism of up to 96% . These models were also used to replicate Hepatitis C and Hepatitis B viruses [72, 73] and screen anti-viral drugs.
Concluding remarks and future outlook
Biomaterials research in liver tissue engineering is exploring a systematic approach of developing purpose-driven biomaterials, engineered cells and constructs tailored for in vitro and in vivo applications. Recent studies have focused on 3 of the 4-component paradigm of tissue engineering: cellular processes [74-76], engineering biomaterials  and cell-scaffold interaction in constructs [78, 79] that employ computational models to quantitatively predict molecular, cell-cell, and cell-matrix interactions. Future research will be extended to the 4th component of the paradigm: integration into living host or in vitro applications. This requires the establishment of quantitative models across all 4 components. Only then, research and development of biomaterials, engineered cells, and constructs would truly be purpose-driven to achieve the design parameters required for the optimal performance in the final applications, even at the onset of the early stage development of the individual components of the tissue engineering paradigm. We envision a coherent marriage of computational and systems biology with biomaterials research in liver tissue engineering [1, 56]. This systematic approach will also be extended to the industrial and manufacturing requirements of the applications so as to accelerate the translational process of bench-side to bed/factory-side.