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International Journal of Bioprinting New challenges in liver tissue engineering

formed by covalent bonds, or physical, which imply to their origin (natural or synthetic), together with the type
non-covalent bonds such as hydrophobic interactions, of cells and the main outcomes of the studies.
hydrogen bonding, chains entanglements, crystallites of
semi-crystalline polymers, or ionic interactions. They 5.1. Natural hydrogels for liver tissue engineering
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can even form dual networks consisting of physical and Liver-derived hydrogels can be created with liver
chemical crosslinks that can sometimes be mechanically decellularized ECM (dECM) since they preserve many
advantageous. Chemical hydrogels are more stable than of the components of the natural matrix that can be
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physical hydrogels in physiological environments in which beneficial for 3D liver cell culture. Willemse et al. used
the pH or ionic strength is subject to changes. They are liver dECM hydrogels to culture and expand intrahepatic
good candidates for liver tissue engineering due to their cholangiocyte organoids (ICO) as alternatives to tumor-
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water sorption capacity and softness in mimicking the derived basement membrane extracts. Liver dECM
liver’s mechanical properties, their ability to mimic the allowed for successful culture of ICOs in dynamic spinner
composition of the liver ECM upon combination, and their flasks, which was more efficient and faster than the
capacity to deliver biomolecules. classical culture in basement membrane extracts. Despite
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the promising results, as the liver ECM varies in different
One of their major disadvantages is that many parts of the organ, the composition of the resulting
crosslinking reactions are cytotoxic, and thus, they are hydrogels is difficult to control, so that chemically defined
not able to be used with cells, although mild reactions hydrogels are preferred in liver tissue engineering, as in
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have been explored; these can take place together with the the case of alginate (Alg), which has been explored in many
cells to form in situ gelling or injectable hydrogels. They tissue engineering applications. In general, Alg has a bad
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are advantageous because the precursor solution can be interaction with cells, and when it is directly crosslinked
mixed with cells and bioactive components and be injected with CaCl salt, it becomes a non-injectable hydrogel due
2
into the body where they become solid, homogeneously to the rapid crosslinking reaction. To make it injectable
entrapping the cells, completely filling a tissue defect and for liver tissue engineering, Tong et al. developed a
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being implantable by minimally invasive procedures. Our two-step method to ionically crosslink the hydrogel with
group has recently optimized a family of enzymatically Ca through an insoluble calcium salt that progressively
2+
gellable hydrogels of gelatin and hyaluronic acid for released the crosslinking ions. To improve the interaction
culturing liver cells. These types of hydrogels can be used with cells, the study used glycyrrhizin triterpene glycoside
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to form 3D construct structures by bioprinting through which provided the hydrogel with specific binding sites for
the development of bioinks (a solution of the precursor hepatic cells. The authors demonstrated that the new GL-
molecules of the hydrogel together with a suspension of Alg-Ca hydrogel had suitable viscoelastic properties for the
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cells). Section 6 of this review contains a summary of the cell culture of hepatic cells (HepG2) with enhanced hepatic
recent advances in this liver tissue engineering technique. functionality and CYP450 expression in three dimensions
The main physical properties of hydrogels, their swelling rather than two dimensions.
capacity, and mechanical properties depend on different Of the chemically defined hydrogels, collagen (Col) is
parameters, including crosslinking density, the chemical probably the best for liver tissue engineering because it is
nature of the polymeric chains (through the Flory– one of the main components of the liver ECM. It forms
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Huggins interaction parameter), the activity of water in the a viscous solution in acetic acid and forms a physical
environment, the concentration of polymers, the porosity hydrogel when neutralized with NaOH. Col bioinks and
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of the hydrogel, etc. The network structure, characterized the resulting hydrogels possess mechanical properties
by its mesh size, determines the diffusivity of water or inadequate for their usage in liver tissue engineering, so
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other soluble species (growth factors or drugs) through they are usually combined with other more rigid polymers,
the hydrogel. The rubber elasticity theory describes the e.g., polycaprolactone grids in the form of scaffolds
mechanical behavior of hydrogels, and the Flory–Huggins built in the melting head of an in-house bioprinter,
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equation describes their equilibrium swelling. The elastic for the heterotypic culture of rat hepatocytes, human
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modulus of hydrogels increases with the crosslinking umbilical vein endothelial cells (HUVEC), and human
density, while the water sorption capacity and diffusivity of lung fibroblasts encapsulated into collagen filaments. The
biomolecules decrease with it. A lower interaction parameter authors described a vascular formation and an enhanced
indicates that more water is absorbed by the hydrogel, and a functionality of hepatocytes (albumin secretion and urea
higher water activity in the environment suggests that more synthesis) in the case of the heterotypic interaction among
water is retained by the hydrogel.
hepatocytes and non-parenchymal cells compared to
Table 1 summarizes the main hydrogels that have been hepatocytes monoculture. Gelatin (Gel), the denaturalized
used for different in vitro applications, classified according version of Col, is an alternative hydrogel widely used in

Volume 10 Issue 3 (2024) 121 doi: 10.36922/ijb.2706