Introduction

The liver is a vital organ in the human body that plays a crucial role in metabolic homeostasis, xenobiotic metabolism, detoxification, and immune regulation [90]. The total number of chronic liver disease cases is estimated at 1.5 billion worldwide, including nonalcoholic fatty liver disease (59%), hepatitis B virus (29%), hepatitis C virus (9%), and alcoholic liver disease (2%) [14]. The hepatitis B and C viruses are the most common etiologic factors for liver cirrhosis and liver carcinoma [5]. The World Health Organization's statistics reported that 354 million people are infected with these viruses in the world [84] . Annually, viral hepatitis, hepatocellular carcinoma, and complications of liver cirrhosis are responsible for approximately two million death worldwide. Hepatocellular carcinoma and cirrhosis were known 11th and 16th-leading causes of death in 2019, respectively [8].

The liver has a unique regeneration capacity, and it can restore mass and function [86]. In critical clinical conditions that the liver couldn't regenerate itself, orthotopic liver transplant (OLT) is the gold standard and known best treatment method for liver failure. However, the limited number of donated livers is one of the most significant obstacles to the broad application of this therapy. Moreover, the use of immunosuppressive drugs causes unwanted side effects and severe microbial infections in these patients.

On the other hand, this surgical procedure causes a high financial burden to the health system and the patient [2]. Another approach is to use extracorporeal liver support devices, which are used for patients on waiting lists for liver transplantation or during the early treatment stages after liver transplantation. The limitations of these devices can refer to restriction in patient's movement, limited processing volumes, the optimum supply of oxygen to cell compounds, and removal of the bile [104]. Tissue engineering could be considered the third modality to reduce the mortality rate due to liver diseases, which does not have these restrictions and is more accessible.

Nowadays, tissue engineering is known promising technology in biomedicine that is developing by using engineering science and biology to repair damaged tissues [4, 48]. TE is the knowledge of new tissue or organ formation from cells, scaffolds, and growth factors. These scaffolds are three-dimensional structures that provide cell growth and a supportive microenvironment for the differentiation and maintenance of cells in ECM [43]. Researchers in tissue engineering science investigate cell implantation by suitable scaffolds, microenvironmental conditions for cell survival, and finally, controlling all factors that contribute to the development of new tissue [34]. In this review, we highlighted natural scaffolds in liver regeneration. In addition, we compared biological scaffolds with synthetic ones used in tissue engineering.

Natural biomaterials

An ideal scaffold should have the following characteristics: biocompatibility, biodegradability with controllable degradation rate, highly porous three-dimensional structure for cell connection, penetration and division to simulate extracellular matrices, suitable mechanical strength to support regeneration, and finally, surface chemistry and topography to improve cellular interactions and tissue growth [67]. Scaffolds are divided into two general groups: synthetic and biological scaffolds. Synthetic polymers are hydrophobic materials with a slow degradation rate in biological conditions due to their high mechanical strength [39]. Many synthetic polymers are used as scaffolds in tissue engineering, and several cases have been specifically designed for skeletal muscle regeneration. Control of physical and chemical properties distinguishes these polymers from natural polymers [115]. These polymers have limitations such as hydrophobicity, low degradation rate, low cell affinity, weak cellular response, acidic byproduct, and unsuitable conditions for drug delivery (H.-S. [62, 64]. The biological scaffolds offer suitable biocompatibility dependent on the material source [44, 83] and do not stimulate the immunity system [3, 103]. These scaffolds can provide environmental conditions similar to extracellular matrices (ECM).

Biological scaffolds are in the spotlight due to the appropriate immune response, mild antigenic properties, angiogenesis, improvement of cellular adherence, and hemostasis [71]. However, natural scaffolds don't support the required mechanical properties of an ideal scaffold for tissue repairing and require modification with other compounds e.g. galactose [33]. Natural scaffolds are usually fabricated in a form such as fiber, powder, 3D printed structures, and sponges. Decellularizing whole tissue or organs also leads to potential natural scaffolds for tissue repair (Fig. 1). In this article, we reviewed the natural platforms used in liver regeneration in three groups: hemostats, hydrogels, and decellularized extracellular matrices (dECM) (Fig. 2).

Fig. 1
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Natural scaffolds shape: fiber, sponge, powder, 3D printed, and decellularized tissue

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Fig. 2
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Comparison of liver reconstruction strategies: A) Hemostat scaffolds inhibit bleeding at the injury or surgery site by absorption and coagulation of blood. B and C) Hepatocytes and endothelial cells are cultured in vitro in the presence or absence of scaffolding, then used in liver regeneration. D) Liver tissue originated from humans or animals is decellularized. Hepatocyte and vascular endothelial cells are perfused into the decellularized scaffold. The repopulated scaffold is placed in a bioreactor to prepare whole tissue and complete the vascular network

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Hemostat-based scaffolds

In recent years, some studies have focused on the integration of hemostatic properties in the studied scaffolds in tissue regeneration. Because these scaffolds, while preventing bleeding at the injuried site, provide conditions for cell proliferation and differentiation. Therefore, in this review article, homeostatic scaffolds are also considered as an independent section.

High blood loss during liver surgeries is a concerning problem [54]. The hemostat scaffolds have a porous structure that provides conditions for fluid crossing from hemostats. Depleting free water stabilizes the hemostat at the injury site and allows tissue repair by absorbing fluids [61, 126]. The high porosity is an ideal property of homeostatic polymer, which is converted to a unique scaffold in tissue engineering. These structures are usually biocompatible, available, biodegradable, lightweight, and low cost [7, 9, 118, 126]. Biodegradability is one of the main parameters that should consider. Otherwise, if the polymers don't decompose on the target site, they must drain from the wound, which can be painful for the patient [61]. Sometimes designed hemostat polymers don't possess highly interconnected porous structures that lead to poor absorption of blood, so inefficient in controlling bleeding [118].

Chitosan is an excellent candidate for hemostat scaffold synthesis due to with antibacterial and hemostasis properties. Some studies have shown that adding a hydrophobic group to chitosan leads to solid hydrophobic interactions with the membrane of platelet, red blood cells, and bacteria that improve its anti-infective and hemostasis strength [16, 25, 26]. Chitosan is not much soluble in physiological conditions. Haddad-Mashadrizeh et al. increased its solubility at neutral pH by blending it with sodium glycerol phosphate [40]. Du et al. designed the alkylated chitosan sponge with microchannels (MACS) that absorbed water and blood. Its shape can be rapidly recovered. MACS has a higher capacity in hemostasis and procoagulant properties than commercial hemostats. In situ experimental results indicated successful vascularization, tissue integration, and infiltration of the liver parenchymal cell (Table 1)[27].

Table 1 Hemostat-based scaffolds for liver regeneration
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Gelatin has benefits similar to other natural materials (Q. [52, 53]. Its most notable feature is activating platelet aggregation that converted it to favorable hemostatic material [66]. Gelatin has a high capacity for blood absorption, which leads to in situ concentrated blood proteins and improved blood clotting [49]. Swelling of gelatin stops bleeding by inducing pressure on the injury site [54]. In recent years, hemostats have been fabricated into forms of sponges, films, webs, silks, and powders [101]. The powder hemostats have always been considered for increasing the contact surface with blood cells [68]. Porous gelatin microspheres (PGMs) showed better hemostatic performance than commercial samples [66]. Fabricated hemostats from gelatin matrix-thrombin, and microbial transglutaminase cross-linked thrombin (TB) loaded Gelatin (mTG/TB/gelatin) reduced clotting time to about one minute [18, 22]. Mushroom tyrosinase and microbial transglutaminase (mTG) were used to crosslink silk fibroin/gelatin that showed a similar result [107]. Three-dimensional gelatin sponges comprised a porous structure with low density, extremely high capacity for absorption of blood and tissue secretions, high surface area, and compressibility, which showed its superiority compared with commercial hemostats, medicine gauze, and gelatin nanofiber membrane (details are shown in Table 1) [116].

Nano or cellulose microfibers have been used widely in tissue engineering [85] and wound healing [108]. Cellulose has a high potential for surface chemical modifications [76] as carboxylation, which improved its absorption and degradation in biological conditions [122]. The blending of oxidized cellulose to the chitosan (CS)/gelatin sponge has improved the hemostatic activity of CS/gelatin (Table 1)[93].

Hydrogel-based scaffolds

Hydrogels are the hydrophobic structure that is insoluble in water. Hydrogels are similar to natural tissue because of their high potential in absorbing a massive amount of biological liquids. In addition, the other advantages of these scaffolds could be referred to as suitable mechanical strength and long life. They have a high potential for carrying cells, drugs, and bioactive compounds. In addition, hydrogels provide an environment for remodeling, migration, and adhesion [79, 99]. Hydrogels keep the wound environment moist and cool and allow oxygen to penetrate, which reduces pain in the patient [124]. Antioxidation, adhesiveness, hemostasis, anti-infection, and drug encapsulation are ideal properties of wound healing hydrogels [36]. Hydrogels structure is impacted by polymer concentration, temperature, pH, cross-linking degree, and salt concentration. These polymers were fabricated by polysaccharides, proteins, synthetic polymers, and their hybrid [79, 99]. Despite the high potential of hydrogels for tissue engineering, their application in medicine has limitations. These limitations include pore size-dependent release rate, limited drug loading capacity, the toxicity of some cross-linker, sensitivity to temperature and pH, low survival of encapsulated cells, and cell damage during photopolymerization [79]. The hydrogels are usually applied for one of the wound types and have limited applicated features [56].

The prepared hybrid hydrogel by Chen et al. significantly improved tissue adhesion, coagulation, hemostasis, and anti-infection in the skin and liver injuries. In addition, the controllable release of VEGF leads to the improvement of cellular proliferation and tissue regeneration (Table 2)[16]. The growth factors have a high affinity for binding to the glycosaminoglycans like heparin, which regulate many of their activities and provide biological stability against proteases activity. The chitosan/galactosylated hyaluronic acid /heparin sponge's ability to carry EGF could improve cell maintenance microenvironment and prolong hepatocyte activities for a long time (Table 2)[32].

Table 2 Hydrogel-based scaffolds for liver regenertion
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Pore size is a limiting factor for cell growth, proliferation, and migration. The photo-cross-linkable gelatin-glycidyl methacrylate was developed from the porous alginate [28] and gelatin methacryloyl (GMA) hydrogels [63], resulting in improving pore size by maintaining mechanical strength (Table 2) [102]. Protease cleavage sites of gelatin lead to its biodegradation during the replacement of new ECM [12]. In addition, the presence of Arg-Gly-Asp sequences gives it the ability to connect to membrane proteins like integrin [70]. However, gelatin-based hydrogels possess weak mechanical and thermal strength as their main limitation [30]. Methylation of lysine residues causes 3D cross-linked polymer formation that is more resistant to temperature [72]. ECM compounds such as hyaluronic acid (HA) can compensate for the poor mechanical strength of GMA [111]. Hyaluronic acid is one of the central compounds of ECM in many tissues. It is responsible for tissue formation, wound healing, and morphogenesis. These linear polysaccharides have a high affinity for adhesive receptors, responsible for poor cell adhesiveness [72, 73]. Cross-linked methacrylic HA (HAMA) improved mechanical and chemical properties. Enhance resistance to enzymatic degradation while maintaining biocompatibility. So far, various hybrids of GMA/HAMA have been made [12, 45, 60], but the combination of these two polymers in a ratio of 4:1 had a significant effect on improving its properties [111]. Chitosan/galactosylated HA (GHA) improved hepatocyte cells adhesion while maintaining liver functions. Added hyaluronic acid to gelatin cryogel increased the elastic modulus while decreasing the scaffold toughness, compressive strength, cell growth, and proliferation (Table 2) [55].

Three-dimensional bioprinting is a high-potential approach for the simulation of ECM in tissue engineering via the building of scaffolds. Hydrogels due to the significant similarity to ECM used as bio-inks [78, 94, 97]. The hydrogels such as elastin, agarose, collagen, chitosan, alginate, gelatin, and hyaluronic acid were used in 3D bioprinting. However, they indicated poor printability [51]. Collagen is the most common compound of ECM used in many bio-scaffolds structures (A. [62, 64]. Despite the benefits of this biomaterial, such as low mechanical strength, high degradation rate, variable elastic modulus, and viscosity during processing, its usage in bioprinting is limited [74]. A slower degradation rate of chitosan than collagen [23] leads to their simultaneous use as bio-inks in bioprinting, improving their degradation rate and mechanical strength [105, 127]. Sue et al. improved the printability of hybrid collagen/chitosan via the formation of hydrogen bonds and careful temperature control during bioprinting. By changing the presence ratio of each, the bioscaffolds properties could benefit different tissue engineering applications (Table 2) [106]. Chang et al. fabricated volvox spheres with alginate and collagen polymers and encapsulated them with MSCs and AML12 hepatocytes. These 3D scaffolds improved liver repair and regeneration in necrosis liver (Table 2) [13]. Reports indicate the unique role of silk-gelatin bio-ink in cell signalings such as Wnt/β-catenin, bone morphogenetic pathways, and Indian hedgehog play an essential role in liver regeneration [98]. Studies have been shown the successful cultivation of hepatocytes into silk scaffolds. Chitosan presence in these structures reduced inflammatory response [100], while RGD peptide improved gene expression [50]. Co-cultivation of hepatocyte and stellate cells in silk scaffolds promoted hepatic functions [114]. Bioengineering porous silk/extracellular protein scaffold provided conditions for co-cultivation of non-parenchymal cells and primary human hepatocytes [59].

Nano-fibrous structures could act as cellular scaffolds due to are their similarity to ECM [113]. Studies have confirmed that nanofibers and the chemical nature of their structure have significant positive effects on cellular proliferation, growth, migration, and adhesion [15]. However, the application of chitosan nanofibers has been limited due to their high degradation rate and edema [21, 123]. The presence of one synthetic polymer such as polycaprolactone (PCL) [41] or polyethylene oxide (PEO) [20] into chitosan nanofibers leads to improved mechanical strength and processability. However, the synthetic polymers, due to high hydrophobicity properties, reduced cell growth and adhesion [10]. Increased hydrophilicity of scaffolds enhances cell growth and proliferation [120]. Studies have indicated that adding hydrophilic compounds like Aloe vera [120], Gum [92], and galactose [37] can enhance hydrophilic groups of scaffolds surface and improve cell adhesion [91].

The importance of 3D shape controlling cellular constructs in tissue engineering led to producing a hepatic lobule-shaped microtissue. These structures were fabricated from capsulated rat liver cells with poly-L-lysine-alginate and have higher hepatic function than spheroids; thus, they could be used as blocks in the bioengineering of liver scaffolds (Table 2) [69]. Yajima et al. simulated a hepatic lobule through assembled cell-laden hydrogel microfiber coated with vascular endothelial cells for liver cells perfusion (Table 2) [117].

Decellularized extracellular matrices-based scaffolds

The biological ECM is prepared from the decellularization of part of the organ or its whole, which its remaining cells made the specific ECM and containing ideal scaffold properties such as complexity, vascular networks, and tissue-specific construction [80, 109]. Stem cells are differentiated based on environmental conditions, which usually don't consider in tissue engineering and tissue repair. Stem cells are often delivered directly into damaged tissue and are ignore tissue biochemistry, needed complexity for 3D scaffold, and vascularity. While tissue repair usually occurs at the wound border, environmental conditions are similar to host tissue [109]. Decellularized ECM (dECM) has tensile strength similar to native tissue and maintains structural integrity. Lack of proper vascular network and primary engraftment transplantation is the most significant limitation in successful tissue engineering [6]. These structures often carry the main vascular branches of origin tissue, which could proliferate and differentiate into the new tissue. Sometimes, they repopulate with endothelial cells or stem cells for new vascular formation [95].

Liver dECM has been studied (Table 3) as a scaffold for proliferation and differentiation of stem cells, endothelial cells, and hepatocytes for the new liver formation and, finally, its transplant (Table 2). Another application of these scaffolds is their use as a liver disease model, which has led to a better understanding of its role in liver fibrosis and discovering new methods for fibrosis therapy by stem cells [57].

Table 3 Decellularized extracellular matrices-based scaffolds for liver regeneration
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The liver-derived ECM was used for liver injuries treatment, but this type of ECM has low mechanical strength [42]. Therefore, the gelatin/PCL/ECM [10] could be a good scaffold in tissue engineering due to the high potential of gelatin in tissue regeneration [128]and the mechanical strength of PCL [77]. Collagen identifies as a central compound of dECM, which leads to in situ clotting. Collagen-coated with heparin in the dECM scaffold was able to give it anticoagulant properties [6]. Also, the heparin-gelatin mixture has been effective in angiogenesis [47]. Anti-endothelial cell antibodies conjugation in dECM causes the proliferation of vascular cells, and it can develop the vascular network in the scaffold structure [58].

Conclusion

Liver transplantation is yet the best option for treating liver failure. However, due to the limited number of liver donors, an alternative method to diminish liver disease mortality is necessary. Over the past few decades, research in the area of xenotransplantation has progressed dramatically. However, cross-species pathogen infectivity and immunological response to the transplanted tissue are the main problems of this type of transplant [19, 35]. Also, traditional cell therapies perfused stem cells directly in vivo, which has led to poor cell engraftment and a low cell viability rate. Hepatocytes function and survival are dependent on cell–cell interaction and connection to ECM compositions. Natural scaffolds can be used as a supportive platform for cell growth, proliferation, and differentiation. Biological scaffolds are biodegradable, decomposing in physiologic conditions, and are replaced with new extracellular matrices. These structures can also simulate cell physiological conditions, which lead to cell growth and differentiation, and finally, liver regeneration. Studies indicated that these scaffolds could simultaneously cultivate two cells type, hepatocytes, and vascular endothelial cells, which caused vascular network formation and finally provided necessary nutrients and oxygen for newly regenerated tissue. Due to their ability to carry drugs and growth factors, they also play a role in regeneration and rescue the liver. Some of them are in a clinical trial for liver disease treatment (Table 4).

Table 4 A review of natural scaffolds clinical trial used in liver disease
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