6
Biomaterials for Liver and Kidney Implants
6.1 Introduction
Transplantation of the liver and kidney is a powerful solution in medicine; thanks to the progress in this field, the number of patients receiving transplants has increased [1]. At the moment, the transplantation represents the gold standard in treating kidney failure and liver disease [2, 3]. However, orthotopic liver transplantation shows significant complications due to infections and organ availability [3]; also extracorporeal devices (like dialysis or artificial liver) are designed for external use and are not able to regenerate the organ [4]. Regarding the kidneys, long‐lasting dialysis is not an ideal solution for patients [5]. Hence, other solutions are necessary and regenerative medicine represents an alternative way through the use of proper biomaterials. The aim of this chapter is to introduce the biomaterials used in tissue engineering for treating liver and kidney diseases.
All the biomaterials used in these implants are designed and developed through the knowledge given by tissue engineering in which several fields of engineering and life sciences converge. The goal of regenerative medicine consists in replacing, repairing, or enhancing the physiological function of a certain damaged tissue (hepatic or renal ones in our case) by finding an equilibrium between synthetic materials, biological compounds, and cellular components [6, 7]. As reported in [8], the engineered tissue is the result of the following:
- Cells: They are the functional unit of the tissue, and thanks to them, the tissue can exploit its functions, and, as a consequence, the organ can work properly. One of the main difficulties that regenerative medicine has to overcome is the use of stem cells; these are capable of self‐renewal and, under certain conditions, they differentiate into the functional unit of the tissue [6]. However, the maintaining and differentiation of these cells are challenging because an adequate cell population is necessary to accomplish the proper physiologic functionalities.
- Growth factors and bioreactor: The environment plays a fundamental role in cell differentiation; indeed the “niches” (the locations of stem cells of a certain tissue) that have been discovered in these years are characterized by several electrical, mechanical, and chemical signals that drive the stem cells to a proper differentiation [9]. This environment has to be replicated through properly designed bioreactors.
- Scaffold: Its role is to provide a template for the cells that can grow into it, thanks to an environment provided by bioreactors. A good scaffold has to respect certain requirements such as biocompatibility: the cell must adhere and proliferate in the matrix; to enhance this process, the architecture of the scaffold has to be well designed. Also, when implanted, the scaffold has to avoid or limit an immune reaction by the organism; biodegradability: during the time, the cells of the host have to replace the matrix; it is important to avoid the release of toxic substances during the degradation process of the matrix; mechanical property: the scaffold has to be adequate for the surgical operation and its role in the tissue; manufacturing technology [8].
All these requisites of the scaffold are strongly dependent on the biomaterials used, which is the focus of this chapter and they will deepen in the following sections. Generally, the scaffolds are made with three kinds of biomaterials like ceramics, synthetic polymers, and natural polymers; the choice of these is driven by the final goal of the implant and the design that has to consider both advantages and disadvantages of the biomaterials used [8].
In the following chapters, the liver and kidney will be briefly described with their pathologies in order to understand their function. With this view, it is possible to understand better the role of the biomaterials used in several applications.
6.2 Liver Biomaterials
6.2.1 General View
The liver is the biggest gland in the body, and it processes blood and secretes bile. It is formed of four lobes, two major ones and two minor ones. It has a double circulation because there are two inlets (hepatic artery and portal vein) and two outlets (hepatic vein and bile duct) (Figures 6.1 and 6.2).
Figure 6.1 Schematics of the (a) anterior and (b) posterior views of the human body.
(From Ref. [3], 2020, Elsevier, CC BY 4.0).
Figure 6.2 Microscopic structure of the liver.
(From Ref. [10], 2018, Elsevier).
Looking at the microscopic anatomy, the functional unit of the liver is the lobule (or acinus). This structure is hexagonal, around 100 microns in dimension, and is repeated million times to form the liver lobes. From its vertexes, the blood flows within the sinusoidal lumen. In this way, the blood can exchange mass with hepatocytes. These cells represent 50–60% of liver cells, and they represent 80% of the liver mass [3]; hepatocytes interact with each other, and they accomplish several roles such as metabolizing substances derived from digestion to derive waste products, metabolizing carbohydrates that are necessary to produce glucose and glycogen (energy reserve), synthesize lipoproteins and cholesterol from lipids, these are used to form cell membranes, synthesize plasma proteins, synthesize bile that participates in the digestive process, detoxify the blood, and metabolize pharmacological drugs. The purified blood is conveyed first in the central vein and then into the hepatic vein, whereas the bile is released into the bile duct of each lobule [11].
The main pathologies of the liver are the chronic liver insufficiency due to the wrong perfusion; the liver loses progressively its functions, and this pathology takes years to develop. Indeed, there is the formation of scar tissue that prevents mass exchange. As a consequence, the blood is not processed properly and toxic substances are carried into several vital organs. The conditions that generate chronic liver insufficiency are hepatitis: viral infections that damage the whole hepatic tissue, intoxication from substances toxic to hepatocytes, hepatic tumors, and cirrhosis; acute liver insufficiency: as opposed to the previous, it can take days or even hours to develop. Necrosis occurs to a large number of hepatocytes, causing a lack of function of the liver. As a consequence, there is an accumulation of ammonia in the blood that reduces permeability and great volumes of liquids accumulate (edema), with consequent damage of the kidneys and the brain. The conditions that cause acute liver insufficiency are fulminant hepatitis, intoxication from substances toxic to hepatocytes.
Depending on the severity of the disease, orthotropic transplant may be necessary; as previously mentioned, other alternatives are cell transplantation, liver support systems, and liver tissue engineering. Hepatic tissue engineering is focusing on optimizing the survival and functionality of the hepatocyte [12]. Indeed, modifications regarding the cell physiology and gene expression occur in isolated primary hepatocytes and also can dedifferentiate into other cell types [3, 12]. This plasticity is controlled by the same cells and the extracellular matrix (ECM) which represents the natural liver scaffold (3% of the total liver area) composed of a complex network of macromolecules like proteins, glycoproteins, or proteoglycans [12]. These interactions between the cells and the matrix have to be considered when designing the scaffold; it has to reproduce the natural environment and also the architecture and stiffness (indeed the right stiffness of the scaffold has to match the stiffness of the host liver [4]).
6.2.2 Biomaterials and Structures
The biomaterials used for liver regeneration belong to three groups: natural polymer, synthetic matrices, and decellularized matrix; these are arranged in several structures such as films, nanofibers, microspheres, hydrogels, and porous scaffold [3].
6.2.2.1 Natural Polymers
Thanks to natural polymers, the scaffold possesses properties similar to the natural structure. The liver cells can recognize them and establish interactions that can vary according to the structure in which they are inseminated. For this reason, natural polymers are used as a coating to attract liver cells and promote their growth [4]. This type of material can be divided between polysaccharides and proteins.
Polysaccharides are macromolecular sugars linked by aldehyde and ketone groups through glycosidic bonds. Hyaluronic acid (HA) is critical for rapid healing and its contribution to the differentiation of hepatic stellate cells (HSCs) into myofibroblasts during the processes of fibrosis has been evaluated. Indeed, if HA macromers are first subjected to polymerization and then to a cross‐linking with UV rays, a more rigid matrix is obtained; in this way, the differentiation is facilitated. Chitosan (CS): It has excellent antibacterial, mucoadhesive (adhesive attachment to a mucus coat), and analgesic properties. Furthermore, its degradation can be controlled by various factors and its waste products are nontoxic. On the other hand, it shows poor bioactivity; for this reason, it is bound with other materials or modified with certain molecules. Several scaffolds made of CS are used in order to maintain hepatocytes in vitro and to promote spheroid formation in hepatocytes [13]. Alginate: It is cross‐linked with divalent ions in order to synthesize hydrogels; it is usually bonded to other materials in order to obtain substrates with certain shapes and mechanical properties. It is used to create implantable constructs, and it increases the functions of hepatocytes and their interaction. Compared to other scaffolds, alginate scaffolds allow for a more vital environment and insemination takes less time [13]. Composite materials: The blends have the strengths of each component. There are several combinations reported by Da Silva et al.; one of these consists of combining agarose scaffold with CS that allows the insemination and growth of hepatocytes in a biocompatible environment. Another example is represented by the synthesis of gelatin with heparin; this material is capable to hold vascular growth factors that allow the proliferation of endothelial cells of the umbilical vein; in this way, the vascularization of the matrix is enhanced.
Proteins are chains of amino acid; among those we can identify: Collagen: It is present in large quantities and allows the matrix to have structural and biological integrity and guarantees dynamism to the structure. Indeed has many cell‐binding motifs, low antigenicity, and high biocompatibility and biodegradability [13]. Although its presence is fundamental, it is unable to provide support to cells and degenerates very easily and in addition is expensive. For these reasons, it is used in combination with other substances in the field of regenerative medicine. Gelatin: It is a biopolymer that is derived from collagen and is soluble in an aqueous environment as it does not have a helix structure (which is present in collagen instead). Through a reaction with tyrosinase and cross‐linking, it was possible to create a matrix with excellent hemostatic capabilities, which are necessary to have an adhesive tissue material. Fibrin: It avoids immunological responses or infections, and once the cells are inseminated, it is replaced with the produced ECM. Although it has these excellent biological properties, it does not have good mechanical characteristics and it degrades easily. More in‐depth studies have shown that, together with a certain type of integrins, fibrin can coordinate the division of hepatocytes during the proliferation phase; moreover, fibrin, fibrinogen, and thrombin scaffolds can resist for adequate times for the growth of hepatocytes.
6.2.2.2 Synthetic Material
As we have seen in the previous paragraph, natural materials have excellent biocompatibility properties but do not possess good mechanical characteristics [3]. For this reason, synthetic materials are used in implants and as drug carriers because of their physical, chemical, and biological properties that are controllable [4, 13].
Aliphatic polyesters are used, and they allow a suitable environment for cell regeneration and remodeling. Generally, these polymers are hydrophobic, so it is necessary to use certain coatings (such as polyvinyl alcohol (PVA) or collagen) or to design certain geometries to facilitate cell binding. Furthermore, due to their hydrolytic degradation, they release acidic substances that lead to inflammation. Usually, aliphatic polyesters are used to build porous scaffolds or nanofibrous membranes in order to allow liver cells to anchor and reproduce [4].
Polylactic acid (PLA), Poly L‐lactic acid (PLLA), Polyglycolic acid (PGA), and Poly L‐glycolic acid (PLGA) (which is the combination of PLA and PGA) are aliphatic polyesters. PLA is a biocompatible and biodegradable polymer; due to its high hydrophobicity, it takes months or years to degrade. PGA supports a functional hepatocyte population [3], and due to its high crystallinity, it degrades in vivo in 2/4 weeks [4]. PLLA and PLGA are biodegradable and are used in implants; their degradation depends on the ratio between crystallinity and molecular weight [13]. They are often used in combination with other materials or they are modified superficially because individually they have had little success, and in these recent years, there is little research on them. According to what reported by Jain et al. [13], a certain combination of these materials has brought certain results: PLGA foams showed properties equivalent to collagen scaffolds; foams composed of PLA, gelatin, polyurethane, and calcium alginate have liver‐specific functions if the foams are used as hepatocyte cultures; inseminations of hepatocytes were more successful in PLGA scaffolds with hydrophilic polymers (Figure 6.3).
Figure 6.3 Scanning electron microscopy (SEM) images of porous PLLA fiber with (a) 1.4 wt%, (b) 1.6 wt%, (c) 1.8 wt%, (d) 2.0 wt%, (e) 2.2 wt%, and (f) 2.4 wt% concentration of polymer in the solution.
(From Ref. [14], Elsevier, CC BY 4.0).
In general, PLLA–PLGA scaffolds are used in bioreactors; although they provide a good environment for hepatocytes to proliferate, PLGA scaffolds, during their degradation, make the environment acidic, causing inflammation [13].
Poly(ε‐caprolactone) (PCL) is another aliphatic polyester; it has a semicrystalline structure, and like the previous ones, it has a good biocompatibility. However, due to its hydrophobicity, it is difficult to inseminate cells [13]; certain proteins are used to facilitate the operation. This aliphatic polyester is also used in combination with other substances to have certain results [3]: by covalently immobilizing galactose on PCL (GPCL), its wettability is increased; this material maintains the physiological functions of the hepatocytes; PCL nanofiber matrices allow the release of certain proteins useful for the regeneration of hepatocytes; according to the study of Bishi et al. [15], a PCL compound (PLACL) was obtained by electrospinning; a scaffold was built with this material and collagen. The result showed that PLACL induces the differentiation of hepatocytes.
As explained above, aliphatic polyesters have considerable limits in tissue engineering; for this reason, other solutions have been found such as polyethylene glycol (PEG), polyacrylamide (PAA), PVA, and elastin‐like polypeptides (ELPs) which are biocompatible and show resistance to the adsorption of proteins that are not specific, and they are electrically neutral and hydrophilic [3].
PEG is usually copolymerized with PLA or PGA; the scaffolds obtained allow to promote the encapsulation of liver cells and promote adhesion and spheroid formation if the hydrogel is equipped with adhesive proteins; furthermore, as reported by Tsang et al. [16], it is possible to maintain the vitality of the cells using photopolymerization.
PAA is used to modulate hepatocyte proliferation and function; indeed, hepatocytes are influenced by the stiffness of the scaffold which can be modified through cross‐linking reactions. This is a very important property because hepatocytes in traditional scaffolds tend to de‐differentiate and only a part of the cultured population develops the necessary functionalities [17].
PVA is able to swell in aqueous solutions without losing its mechanical characteristics, thanks to its crystal lattice; in the studies by Bidault et al. [18], fibrin hydrogel was associated with PVA inside an interpenetrating polymer network (IPN) architecture and the fibrin was preserved and maintained its mechanical and hemostatic properties. IPN is defined as a combination of two polymer networks cross‐linked in the presence of the other; this structure was used in another porous scaffold with CS and a copolymer made of acrylonitrile (AN) and N‐vinyl‐2‐pyrrolidone (PANNVP). In this case, thanks to the copolymer, the adhesion and functionality of the hepatocytes were increased and the CS favored their encapsulation [19].
ELPs are often used in tissue engineering applications as it is possible to control the chemical and physical characteristics of the material and, consequently, to control cellular functions. In the case of the hepatocytes, these have had a large expansion in scaffolds consisting only of ELP or ELP conjugated with negatively charged polyelectrolytes (polymers in which a charged monomer subunit is repeated) has promoted [20].
6.2.2.3 Decellularized Matrix
Decellularization consists of removing the cells from the tissue so that only the ECM remains [3]. In this way, it is possible to use a natural structure with a certain architecture and vascularization that allows an efficient recellularization [13]. In the previous examples, we have seen different types of biomaterials that allow the construction of a scaffold through a bottom‐up approach. On the other hand, with decellularized matrices, a top‐down approach is used; decellularization can be faced with physical, enzymatic, or chemical methods. The most efficient is the use of detergents and ionic salt solutions. Thanks to their composition and nano‐micrometric structure, these matrices represent excellent scaffolds; however, they are still used little for applications on humans due to their little availability [4].
In the study by Skardal et al. [21], it can be observed how the survival and functions of human primary hepatocytes are maintained and improved through the use of hydrogels consisting of decellularized matrices with natural polymers (e.g. collagen), hydroxyapatite (HA), or heparin conjugated. Such constructs can also be used for toxicological analysis.
However, it is necessary to underline that the vasculature of the decellularized matrices does not allow a physical exchange of nutrients and metabolites and this causes the degeneration of the cultured cells. For this reason, human umbilical vein endothelial cells (HUVECs) were used in the scaffold obtained described by Shirakigawa et al. [22], which allowed a more developed vascularization; this resulted in the development of hepatocytes around the vessels.
Another problem that can be encountered in decellularized matrices is thrombogenicity, which, as shown in the study by Bruinsma et al. [23], can be attenuated by a layer‐by‐layer deposition of heparin in the matrix without affecting recellularization. This study was performed on rat liver tissues, so similar considerations cannot be made with human livers.
Structure
As mentioned in the Introduction, cell regeneration is also determined by the scaffold structure; for this reason, the biomaterials assume certain geometries. The most used structures are porous scaffolds, nanofibers, hydrogels, and microspheres.
Porous scaffolds
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