2. Biomimetic Hybrid Systems Based on Natural Polymers for Tissue Engineering

Tissue engineering applications require the use of complex scaffolds where an accurate selection of materials is essential. In addition, it is advantageous that these materials could accommodate growth factors and cells, while providing cues to guide cell adhesion and proliferation [9,10].

Hydrogel scaffolds appear ideal matrices to culture cells and produce in vitro tissues [11]. Probably, polysaccharides (Figure 1) constitute the most interesting family of polymers due to their high hydrophilicity, their origin linked to the living tissues, their biodegradable and biocompatible properties, their ability to render hydrogels with similar properties to ECM [12], and their capacity to display bio-responsive functions [13]. Nevertheless, chemical modifications appear necessary to facilitate the attachment of cells, which may be a problematic feature since a complete removal of employed toxic agents is essential for subsequent biomedical applications [14]. Direct blending of polysaccharides is a recent strategy that is applied to improve cellular adhesion and proliferation as recently reviewed by Ng et al. [9].

Figure 1. Natural polysaccharides able to produce hydrogels and respective sources. Reproduced with permission from [9].

Cellulose and chitosan are between the most employed hydrogels for biomedical applications. In fact, they are the most abundant natural polymers in earth. Cellulose appears an ideal matrix for tissue engineering applications [15], alone or even blended [16] with other polymers like chitosan. Hydrogels can be prepared by crosslinking of aqueous cellulose esters [17], a procedure that avoids the limitations caused by insolubility of natural cellulose in aqueous media. Nanocellulose (i.e., cellulose nanocrystals [18], CNCs, cellulose nanofibers [19], NFCs, and bacterial cellulose [20], BC) can also be incorporated into polymer matrices to improve mechanical properties. For example, the interpenetrating network of gelatine and alginate reinforced with CNCs can improve the performance of natural cartilage, favor cell adhesion, and protect cells from immune rejection [21]. A second interesting example about the use of nanocellulose corresponds to the development of BC membranes coated with alginate and collagen in each side [22].

Deacetylation of chitin leads to chitosan, which has a great structural stability, capability to absorb water, and a cationic character that favors the formation of gel particles through electrostatic interactions [23]. Chitosan is mostly employed as injectable hydrogels, crosslinking agents like genipin [24] and even hydroxyapatite particles being added to improve osteogenic properties and enhance cell adhesion and proliferation [25].

Collagen is the most relevant structural protein in the human body [26] and is the major component of the native ECM. Therefore, great efforts have been focused on preparing collagen-based scaffolds to mimic native environment [27]. Solution processing (e.g., electrospinning) of collagen has some restrictions since solvents used to denaturalize the highly insoluble triple helix structure (e.g., 1,1,1,3,3,3-hexafluoroisopropanol) should be avoided due to their toxicity [28]. To this end, co-electrospinning has been proposed using a biodegradable synthetic polymer able to provide elasticity (e.g., the copolymer of l-lactide and ε-caprolactone) and a mixture of collagen and a water-soluble sacrificing polymer (e.g., PVP) that acts as a transporter polymer for an easy spinning of collagen [29].

Different scaffolds have successfully been developed to mimic the 3D organization of interstitial ECM, but scarce results have been reported concerning the reproducibility of the 2D ECM basement membrane (BM) [30,31]. These membranes establish functional polarization of epithelial and endothelial cell layers throughout the entire body [32,33] and appear fundamental for artificial organ technologies [30,34]. Furthermore, BMs assure tissue compartmentalization and may prevent the spreading of cancer cells [35]. Basically, BMs are based on the assembly of two different proteins: type IV collagen (Col IV) and laminin (LM), which provides unique mechanical properties for an effective protection of tissues from external stresses [36]. Artificial matrices mimicking BMs have been prepared by co-assembling polylaminine and Col CV under acidic conditions. A layered morphology (thickness around 15 m) was derived, its great capacity to support keratinocytes and form a cell layer close to the architecture of natural epidermis being demonstrated [31].

Gelatine, as a soluble denaturalized collagen, is widely employed for tissue regeneration due its relevant role in cellular metabolism and morphogenesis. Moreover, gelatine is interesting for giving rise to thermosensitive scaffolds taking into advantage its capability to form solid gels at low temperature through intermolecular hydrogen bonding interactions [37], which can be disturbed at increasing temperatures to form liquid gels.

Mixtures of gelatine with other natural polymers like agar and alginate have also been evaluated. Thus, agar–gelatine mixtures have been found appropriate to produce hydrogel scaffolds with a uniform internal pore structure [38], and gelatine/alginate mixtures were found suitable for manufacturing biological scaffolds by 3D bioprinting [39]. Natural gelatine has also been blended with synthetic polymers such as nylon 6 and polyurethane to render suitable scaffolds for bone tissue engineering [40]. New scaffolds facilitated apatite-like mineral deposition, and promoted osteoblast cell attachment, migration, and proliferation [41]. Hybrid nanofibrous scaffolds have also been prepared by layering a poly(3HB-co-4HB) copolymer and gelatine [42]. The trilayered system with gelatine in the middle showed a good water-resistant ability and high proliferative activity of mouse fibroblasts. Hydrogels based on gelatine and methacrylamide have been found appropriate to simulate liver tissue, a hepatocyte cell survival rate of 97% being found [43]. It can be deduced that hybrid composite materials with natural and synthetic polymers are gaining relevance to develop biomimetic scaffolds [44,45,46].

Fibrin, keratin, alginate, and hyaluronic acid are other relevant natural polymers that are gaining relevance for the development of biomimetic scaffolds. Fibrin is a structural protein employed to prepare bioprinted scaffolds [47]. Specifically, scaffolds with embedded neurons have been, for example, prepared by a layer-by-layer printing process These materials can attain modulus and tensile strength of 2.9 and 1.7 MPa, respectively [48]. The fast gelation rate of fibrin can be combined also with the high biocompatibility of collagen to render scaffolds with fast wound closure and high vascularization [49].

Keratin is a structural fibrous protein that forms part of epidermal structures (e.g., nails, wool, hair, and feathers) and provides toughness and resilience depending on the number of sulfur cross-links that are established through their cysteine units [50,51]. Keratin can be extracted from natural materials by different processes such as the use of ionic liquids [52], microwave irradiation [53], alkaline treatment [54], and reduction of disulphide linkage [55]. Capability of keratin to self-assemble and support cellular proliferation enhance its use in tissue regeneration applications [50,56]. Between them, the reconstruction or regeneration of ocular surface [57], urinary track [58], and nerves [59] are significant. The relatively poor mechanical properties and brittle structure of keratin justified its blending with different synthetic (e.g., poly(ε-caprolactone (PCL), polylactide (PLA), polyvinyl alcohol, and polyethylene oxide) and natural (e.g., chitosan, gelatine, silk, fibrin, and hyaluronic acid) polymers [51].

Sodium alginate is a natural polysaccharide widely employed in tissue engineering despite some limitations like low mechanical integrity to form suitable fibers and too high biodegradation rate to be used as a supporting matrix. Nevertheless, this polysaccharide has several advantages for cartilage tissue engineering as recently reviewed [60].

Hyaluronic acid (HA) is a glycosaminoglycan that can give rise to fibrillar structures with good mechanical performance and a regulatory function [61]. HA has interest in regeneration processes [62] and consequently is considered in tissue engineering applications. Fabrication of HA scaffolds with high porosity (i.e., 70–95%) and swelling ratio (i.e., 2000–6000%) has been reported. These materials display soft-tissue mimetic mechanical properties (≈0.5–1.5 kPa), accelerated tissue formation, neovascularization, and reepithelialization in vivo [63].

Interest towards peptide hydrogels is emerging despite their higher cost with respect to typical polysaccharide-based materials. Efforts are logically focused to mimic the natural fibrillar proteins of ECM by means of synthetic peptides able to self-assemble. Different morphologies are derived depending on the peptide sequence. Basically, strategies to favor self-assembly are based on (a) an alternate disposition of hydrophilic and hydrophobic residues (e.g., Arg-Ala-Asp-Ala characteristic of the named RADA peptides) [64]; (b) the use of complementary peptides (i.e., peptides having opposite electric charges) [65]; (c) peptide amphiphiles [66]; (d) cyclic peptides [67]; (e) functionalized peptides [68] (Figure 2). High cost, non-renewability, complex purification processes, and demanding storage conditions are some drawbacks that limited the use of protein-based hydrogels despite their inherent cell adhesivity [69,70].

Figure 2. Strategies developed to favor peptide self-assembly: alternate disposition of hydrophilic and hydrophobic residues, complementary co-assembling peptides, peptide amphiphiles, cyclic peptides, and functionalized peptides. Reproduced with permission from [70].

Electrostimulation (ES) has beneficial effects on tissue regeneration (i.e., muscle, bone, skin, nerve, tendons, and ligaments) since it enhances cell proliferation, ECM synthesis, cytokines production, and vasculature development [71]. The development of scaffolds based on electroactive polymers is highly interesting to facilitate the successful application of ES. Conductive polymers (i.e., based on electronic conductivity enabled through oxidized pi-bond conjugation states) are interesting but have poor properties (in general are brittle and non-degradable thermosets with a conductivity that decreases progressively as a reduced state is attained). Usually, composite systems based on conductive polymers like polypyrrole, polyaniline, and polythiophene derivatives, and natural polymers like chitosan, cellulose, and alginate have been selected. Alternatively, the incorporation of ionic functional groups (e.g., carboxylic, sulfonic, and amine) to a polymer can generate ionic conductive materials without modifying significantly biocompatibility and degradation profiles [72].

Incorporation of carbon nanotubes promote cell proliferation and facilitate nerve regeneration. Conductive hydrogels have been prepared from N-isopropylacrylamide (NIPAM), laponite, and CNTs [73], also being remarkable the derived improvement of mechanical properties. This characteristic was considered to prepare hybrid hydrogels constituted by gelatine, sodium alginate, and CNTs to be employed as bioink to print hollow tubular scaffolds. These led to blood vessels after colonization by epidermal fibroblasts. Doping with CNTs was essential to enhance mechanical properties while no cytotoxicity was found [39].