The significance of biomacromolecule alginate for the 3D printing of hydrogels for biomedical applications

Abstract

Natural biopolymers have been widely employed as biomaterial ink hydrogels for three-dimensional (3D) extrusion bioprinting in the preparation of the next generation of bioengineering materials for healthcare applications. Alginate is a linear anionic polysaccharide with favourable properties, such as: typical rheological (gelling, viscosifying, and stabilizing dispersions) characteristics, biodegradability and biocompatibility properties. However, in order to improve alginate applicability for practical biomaterial/bio ink for advanced medical applications, it is often modified and functionalized with several polymers and nanomaterials in order to obtain better printability of alginate-based biomaterial/bio ink hydrogels. This review, principally, emphasizes the recent developments and with a comprehensive overview of alginate-based biomaterial/bio ink hydrogels and their biomaterials (3D scaffolds, tissue-like structures with hierarchical vasculatures, mimics of biological, physiological and pathological functionalities) for biomedical applications. It also addresses the significance of alginates, oxidized alginate and their functionalizations (interface) with various materials in order to improve the biomaterial/bio ink properties for 3D extrusion bioprinting applications. Finally, it provides current advances, vital roles and new perspectives of alginate-based materials and their future developments for 3D bioprinting purposes.

1. Introduction

In the last few decades, tissue regeneration or organ replacement in healthcare, has increased significantly, worldwide [1]. However, in order to achieve healthcare requirements, effective 3D bioprinting technology is needed to generate advanced artificial tissue or biomaterials for use in biomedical applications/procedures and tissue engineering [2]. In addition, the development of suitable tissue or comparable tissue biomaterials, depends mainly, on biomaterial ink/bio ink hydrogel and its composition [2]. Bio ink has a gel form that can be prepared with several biomaterials, including living cells, whereas biomaterial ink also has multi-materials, except for cells and it is used for printing [3,4], thereafter, the printing of biomaterials that can be used for cell encapsulation [5]. Primarily, biopolymers have been used to form a novel ink in order to design tissue engineering biomaterials by using several types of 3D printing methods, such as: Extrusion, Inkjet-based technique, Pressure-assisted technique, the Laser-assisted bioprinting and the stereolithography [6].

In this technology, the extrusion of 3D bioprinting has been used to print three-dimensional biomaterials (layer-by-layer) for tissue engineering. This is due to its ease of operation, affordability and capability to bioprint high cell densities and other features, such as: a nozzle, a syringe and a pressure system [7,8]. In addition, when compared to other bioprinting methods, extrusion-based bioprinting can print high viscous bio-inks [8,9]. Moreover, cells can incorporate directly, several extrusion bioprinters that have multiple print heads in order to print advanced multifunctional biomaterials. These key features make them popular in medical sciences. However, the 3D bioprinting technology can offer unprecedented versatility in biomedical applications, needed to reproduce suitable native tissues by using biomaterial/bio-ink [10]. Furthermore, this technology is a computer-controlled process that provides rapid design and fabrication of complex structures without expensive tools [11]. However, biomaterial ink should have noble biocompatibility, suitable rheological and fast cross-linking ability to retain the 3D structure after printing. These characteristics offer a unique hydrogel property that absorbs and retains a large amount of water (Supplementary Table 1) and maintains its stable form, under the biological milieu.

Ionic interactions, hydrogen bonding, impurities (additive) and stacking are the characteristics of supramolecular interactions with biopolymer chains that result in combinations of the associated materials. These requirements indicate that supramolecular interactions (weaker than covalent interactions), should speed-up bioprinter production, improve the surface structure for managing cell growth/scaffold interactions and impart the necessary targeted mechanical characteristics by strengthening the required interlayer adhesion [12]. In addition, natural polymers have immune-modulatory characteristics, which can play a significant role in the physiological processes, necessary primarily, for amicable healthcare applications [13]. However, according to reports, biopolymer supramolecular functionality, is enhanced by the modification and functionalization with small macromolecules, including their decoration with nano-/biomaterials [14,15]. Therefore, these materials can create significant supramolecular interactions (ionic, guest/host, hydrogen, π-π) between polymer chains in the ink and provide dynamic structure (reversibility of interactions) and tunable viscoelastic behaviour [12]. Therefore, several biopolymers (H-bonding and ionic) interactions or orthogonal functionality can change the flow behaviour of biomaterial/bio ink.

Initially, in the stage/process of development of biomaterial ink, a single biomaterial, a simple physical mixture of single materials and single cell type materials were used as ink for the 3D bioprinting process. However, the single ink usually cannot meet all the mechanical and functional requirements and these are essential in order to produce biomimetic tissue-like constructs. For example, the single polymer (i.e., polyethylene glycol, gelatin and fibrin) bionks, have a few disadvantages, such as: cell adhesion, proliferation and spreading and mechanical properties [16,17]. Of recent, there have been useful strides made to improve the biomaterials ink properties that can be developed from more than one polymer with other bioactive components [18,19]. As a result, the improved performance of the biomaterial-ink has become more attractive for 3D bioprinting of constructs [20]. However, suitable biomaterial inks should have a storage modulus of between 10² and 10³ Pa and a viscosity of between 30 to >6 × 10⁷ mPa/s is appropriate for the extrusion of 3D bioprinting [21,22]. If these dynamic viscosity values are at low shear rates, i.e., around or below 1 s⁻¹, since anything below 1 Pa·s (10³ mPa·s) is a low viscosity liquid and hence, it is completely unsuitable for the extrusion of 3D bioprinting. On the other hand, if the values are at a high shear rate, close to those typically achieved during extrusion bioprinting, i.e., around 10³ s⁻¹, or anything above 10 Pa·s (10⁴ mPa·s), it is considered to be an extremely high viscosity and it is incompatible with the presence of cells within the hydrogel. In addition, in order to print these biomaterials/bio ink by using the extrusion 3D bioprinters method, there is a need for the process and the printer (temperature) characteristics to be optimized in order to avoid cell damage in the bio ink. According to Sanchez et al. report, the cartridge (where the inks are loaded) temperature or printing temperature of between 20 and 40 °C is adequate, depending on the materials. In contrast, the biomaterials ink’s low viscosity that induces less shear stress and hence, decreases cell damage, must be thoughtfully considered [23]. The cartridge temperature is usually between 30 and 40 °C and it is often used for cell-laden hydrogels. In early literature, the combination of alginate, gelatin, agarose or GelMA with cell incubation temperature (37 °C), has been reported [24].

However, to achieve a homogeneous cell-distributed biomaterial, depends mainly, on the mixing process of the bio ink. During this process, cell death occurs due to the mechanical impact on the cells. The specific mechanical effects depend on the rheological characteristics of the bio ink. The high shear stress causes cell death during extrusion. Therefore, the rheological characteristics of ink and the bioprinter characteristics (mainly, nozzle diameter) need to be optimized to minimize cell death. Lastly, researchers have reported the ionic crosslinking step (of biomaterial) that leads to cell death, while others have not found evidence of significant cell death [25]. None of these effects are related to the (or lack of) cytotoxicity of the alginate or the incubation temperature, following the printing. However, optimized biomaterials/bio ink is needed to print advanced biomaterials by using extrusion 3D bioprinters under specific conditions.

In general, for the individual biomaterials, the ink should have enough strength to enable the engineering of mechanically demanding tissues, such as: bone, cartilage and tendon. These properties may be needed to adjust the strength of bioprinting hydrogel by combining them with natural biopolymer in order to obtain functional multicomponent-based biomaterials/bio ink. However, positively charged polysaccharides can provide an inflammatory response to the hydrogels [26]. In order to overcome these concerns/issues, positively charged polymers are combined with negatively charged polymers to prepare active bio ink [26]. Therefore, negatively charged natural polymers are highly desirable to enhance the ink properties, such as biocompatibility and cell viability [27,28].

Alginate is a biopolymer and it has a favourable (α-L-guluronic acids) functionality for structural modifications. It offers accessible physical cross-linking in the presence of non-toxic divalent cations and metal ions via electrostatic, ionic interactions, covalent bonding, redox reactions and coordination chemistry [29]. In addition, alginate and modified alginate are used in various industrial applications (Tissue engineering, Scaffolds for drug delivery, Biocidal materials and Implant materials) because of their rheological characteristics [30]. Such features are mainly attributed to its functionality [α-L-guluronic (G) and 1–4-β-D-mannuronic acid (M) monomers] [31,32]. The alginates are unbranched binary copolymers, linked by M and G residues. In addition, they are considered as block copolymers, consisting of orthomopolymeric regions of M and G, termed M-and G-blocks, respectively, interspersed with areas of (MG) block (Fig. 1a–c). In the coordination and chelating structures in the model of an egg-box, during the process of binding alginates to polyvalent metal ions, the G-units selectively form higher-order junction zones, which are composed of two or more chains, together with the hydrogen-bonding interaction of these cross-linking agents with oxygen atoms in the G blocks of two adjacent polymer chains (Fig. 1d). The unique properties of alginate are its: biological origin, non-toxicity, hydrophilicity, biocompatibility, biodegradability and non-inflammatory response. In addition, the alginate hydrogel crosslinked by Ca²⁺, has the stress relaxation property (elastic behaviour; it can regulate cell spreading and proliferation), which is like the human extracellular matrices. These remarkable properties facilitate their suitability in many clinical and biomedical applications. Therefore, it can be widely used to form biomaterial ink for extrusion of 3D bioprinting applications and improve the biocapacity of the biomaterials [33]. Due to its good tissue compatibility, it has been widely used in tissue engineering, including regeneration of skin, cartilage, bone and liver, to treat exuding wounds and enhance the healing process [29]. The hydrophilic nature of alginate, makes it possible to blend and cross-link with other materials, such as: organic and inorganic materials, which enables the preparation of new biomaterials [34,35]. According to recent reports, a high M-block content of alginate is helpful for biomedical applications due to the alginate’s capacity to encourage good cytokine production via human monocytes [29]. In summary, alginate and other natural polymers have been used to form a novel biomaterial/bio ink, useful to design hydrogels by using several extrusion 3D bioprinting methods (extrusion, inkjet, laser-assisted bioprinting and stereolithography) [36].

Fig. 1.

Structural characteristics of alginate (a) alginate monomers, (b) chain conformation, (c) block distribution [37]. Copyright © 2005, John Wiley and Sons and (d) The junction zone in the egg-box model of Ca²⁺ ions alginate gel [27] (Open Access This article is licensed under a Creative Commons Attribution License, MDPI).

This review article elaborates the importance of alginate to the formation of biomaterial/bio ink (with nanoparticles and other polymers) for the development of biomaterials in extrusion 3D bioprinting technology. Additionally, recent (last five years) advances, the significant roles and new perspectives of alginate-based biomaterial/bio ink hydrogels for future developments in extrusion 3D bioprinting, are succinctly discussed.

2. Biomaterial/bio ink characteristics

Principally, biomaterial/bio ink is an aqueous mixture of multi-components (Supplementary Table II) material that is formed through physical (reversible interactions: hydrogen bond, ionic and van der Waals) or chemical (irreversible: covalent) hydrogels (Fig. 2). According to recent reports, biomaterial/bio inks should be biocompatible for tissue engineering applications and they should have good biodegrad-ability, bio-printability, sufficient viscoelasticity, in-situ gelations, shear-thinning (lower viscosity) and permeability (of nutrients and gases to improve the metabolic activity) characteristics [31]. Biomaterial inks can also interact with cells and enable encapsulation property at a pH of between 6.5 and 7.8 and printable between 5 and 40 °C (cell survival) with slight pressure (100–200 kPa to reduce cell stress) in order to minimize cell death [40]. In summary, in order to prevent specific conditions for the production of ink, the first reason for cell development temperature should not exceed >40 °C–5 °C<. Higher and lower temperatures are caused by the living environment of the cells, which causes cell death or growth retardation. The second issue is that a change in the pH (>6–8<) of ink can minimize the viability of the cells and worsen the cell morphology [41]. Therefore, suitable (needed for the optimization of pH) ink should be used for cells loading and to be used to print under specific conditions. Additionally, biomaterial/bio ink, prepared with non-toxic catalysts, cross-linkers and solvents are beneficial for biomedical applications. The shear-thinning property is an essential parameter for forming alginate-based bio ink for extrusion bioprinting [42]. The alginate-based ink has been used for extrusion 3D bioprinting for tissue-engineered applications. However, due to the shear-thinning behaviour of alginate ink, its viscosity decreases (lower viscosity provides less shear stress, hence, decreasing cell damage), which can cause difficulty in printing when using the extrusion 3D bioprinting technique. In order to overcome this problem, the in-situ cross-linking limitations are improved by using various components.

Fig. 2.

Composition of biomaterial ink to extrusion 3D bioprinting for the design of the next generation of bioengineering materials for healthcare.

Additionally, oxidized alginate can control the degradation behaviour of ink and improve the shape fidelity of 3D printed biomaterials. Lately, the shape fidelity of alginate ink was improved by adding CaCℓ₂ solution and support materials, e.g., cellulose nanocrystal [43]. However, biomaterial/bio ink formation mainly depends on the target (tissue, organs, drug delivery system) scaffold and the printer used (Fig. 2). Biomaterials can be printed and then seeded with cells after printing; however, they are not directly made with cells that are generally, not considered as bio inks. As a result, the phrases are referred to as biomaterial inks [40]. In bio ink, cells are the main components (Supplementary Table III) and they are combined with polymers, bioactive components and other supporting materials and they can be used to fabricate biomaterials (via biofrabrication methods).

2.1. Rheology and printability

The biomaterial/bio ink’s rheological behaviour plays a key role in its printability. The rheological characteristics can be used to study the deformation and flow behaviour (viscosity, shear-thinning, yield stress and shear recovery) of biomaterial/bio inks under the applied forces [44]. In addition, these rheological properties help to improve the printability of ink (Supplementary Fig. 1), while maintaining cell viability [45]. The viscosity of the ink is a measure of its resistance to flow under shear stress [45]. Knowing the ink’s viscosity properties is essential in a 3D printing technology because they relate to the flow of the materials as it is spread via the printing nozzle. The high viscosity ink has high printing fidelity and high viscosity leads to increased shear stress that can affect the cell that is suspended in the ink [46]. Therefore, low viscosity ink is suitable for cell viability [47].

Generally, polymer-based ink (solutions or blends) exhibits shear thinning or shear-thinning behaviour [48]. This behaviour is known as non-Newtonian behaviour that can be determined from the flow curves, drawn from the viscosity versus shear rate [49]. The ink viscosity increases with increasing shear rate, which means that the ink exhibits shear-thickening behaviour, which is irrelevant for 3D printing applications. Conversely, the shear-thinning behaviour is exhibited if the ink viscosity decreases with increasing shear stress. The shear-thinning behaviour is essential for all the bio inks for the survival of the cells [44]. However, the shear-thinning behaviour is required for the ink during the printing time. Due to this behaviour, ink can flow easily from the nozzle when there are increases in the shear forces.

The yield stress of bio ink is characterized as the minimal stress required to induce flow in the ink and the ink flow can be measured via the shear rate or shear stress frequency sweeps and the fitting of the flow curves [45,50,51]. However, the yield stress of the ink is determined via the plotting of the viscosity versus the shear stress [50]. According to reports, the high yield stress of an ink can improve its printability [50]. It is also essential to obtain an ideal ink for extrusion-based printing [52]. Additionally, the biomaterials/bio ink should exhibit good shear recovery (stop flow and ability to recover structural stability) properties, after the printing, within seconds. The bio ink elastic recovery (solid-like) behaviour is simulated via oscillatory time sweeps with high and low shear stresses at a constant frequency, in order to quantify the elastic and viscous moduli [53]. This viscous flow and elastic recovery behaviours is known as viscoelasticity [46,48]. This behaviour can be expressed by utilizing two parameters, viz: i) shear storage or elastic modulus (G′) measures the stored energy and expresses the solid-like character of the inks and ii) shear loss or viscous modulus (G″) measures the amount of energy dissipated by the bio ink and expresses the viscous flow, which can permit for cell encapsulation and extrusion. However, the biomaterial/bio ink flow behaviour is highly dependent on the molecular weight of the polymers and other components [48]. Therefore, the biomaterial/bio ink rheology and printing parameters consistently, vary. Jessop et al. specified that a 0.625% of alginate ink, exhibited superior properties, but quickly collapsed at room temperature, post-printing [54]. Concentrations of 7.5 and 10% alginate ink, have high viscosity and they make it difficult to introduce nanocellulose. Concentrations of 1.25, 2.5 and 5% of alginate-based inks, have promising viscosities and other materials can be introduced into these inks. Finally, the authors reported that a 2.5% of ink has better rheological properties and it is suitable for 3D printing applications.

3. Influence of alginate molecular weight and concentration on printability and cellular functions

Viscosity, cell viability (migration, proliferation), degradation and other physical properties of biomaterial inks and biomaterials are mainly dependent on the alginate molecular weight (20–400 kDa) and concentration [55]. According to reports, low molecular weight alginate (28 kDa) has proved to be less viscous to the biomaterials ink and such class of alginates requires ~2.5 times more cross-linker to form biomaterials when compared to the high molecular alginates (75 kDa) because low viscosity bio inks are not stable after extrusion via 3D bioprinting and they require effective cross-likers in order to obtain the final product [56]. Similarly, a low amount of alginate, provides low viscosity bio ink than a high amount of alginate-based bio ink (high viscosity) [57]. Therefore, alginate concentration can modify the viscosity of biomaterials ink and based on its viscosity, it can be used for extrusion in 3D bioprinting applications. Generally, a high viscosity (calculated from the shear stress and the shear rate) bio ink, needs high shear stress during the printing process, which is not suitable for cells encapsulation in a bio ink [58]. However, if the viscosity of the bio ink decreases, this means that its shear rate will increase and hence, reduces the shear stress (shear-thinning behaviour) and improves the cell viability [59]. In addition, the low viscosity bio ink can reduce the nozzle blockage in different 3D bioprinting techniques. Therefore, low viscosity (shear stress), exhibited significant cell viability, non-uniform cell distribution and aggregation due to the faster sedimentation when compared to the higher alginate concentration [60]. In addition, the low molecular weight-based biomaterials (hydrogels) are quickly degraded in a phosphate buffer saline when compared to the higher alginate concentration. The different molecular weights of alginates can control the alginate hydrogels’ degradation behaviour and drug delivery/growth factor delivery from the hydrogels [61].

The alginate-based biomaterial/bio ink printability also depends significantly on its molecular weight and can be used as ionic crosslinkers [56]. Naturally, when low molecular weight alginate is used in ink, its printability is easier than the high molecular weight alginate, used in ink; this is because it needs a low amount of crosslinker to print hydrogels [62]. Therefore, the selections of alginate molecular weight and its concentration are important for the suitability of biomaterials/bio ink and the formation of biomaterials for advanced in-vivo and in-vitro biomedical applications.

4. Material property with microscopy analysis for alginate-based systems

The 3D printing technologies can easily form macro/microporous biomaterials via a controlled programming with an extrusion process. The porous nature can allow cell migration, adequate nutrient supply and tissue formation [63,64]. Wattanaanek et al. developed biomaterial inks from alginate, cellulose and amorphous calcium phosphate/calcium sulfate hemihydrate [63]. The inks developed were used to print porous scaffolds for bone regeneration. However, in order to create the porous nature in the alginate biomaterials, amorphous calcium phosphate (ACP) and calcium sulfate hemihydrate (CSH) are used. Their pore size (298 to 377 μm) was observed with scanning electron microscopy. In addition, the 18% and 20% ACP/CSH biomaterials, exhibited the highest cell proliferation. Finally, they concluded that 20% of ACP/CSH-based alginate materials are better suitable for bone cell regeneration.

Somasekaran et al. developed a bio ink from alginate, gelatin and diethylaminoethyl cellulose [64]. The ink developed was used in the preparation/fabrication of a porous skin tissue. They specified that diethylaminoethyl cellulose can improve the cell viability, stability and also the porosity of the hydrogel. The microstructure of the hydrogel was observed from scanning electron microscopy (SEM) and the micro-computer tomography (Micro-CT 40). In the Micro-CT, they observed small and big pores as confirmed by SEM. From the SEM, they noticed that the smaller pore sizes were between 90 and 150 μm and the bigger pores were from 500 to 700 μm. However, the small pores are helpful for cell mobility and the bigger pores are helpful to allow nutrients and oxygen. Jessop et al. used the transmission electron microscopy to analyze the alginate-nanofibrils bio ink morphology (porous) and the crosslinking effect of CaCℓ₂ [54].

5. Alginate-based biomaterial/bio ink

Alginate is an anionic biopolymer that enables the preparation of hydrogels for biomedical applications because it is a proven biocompatible, porous (easy transport of fluids) and hydrophilic natural biomaterial [65]. However, alginates are usually restricted under mechanical loading, owing to their brittleness and low elasticity [66]. In addition, alginate hydrogels have unstable swelling, degradation and poor adhesion properties due to a lack of functionality and cell-binding domains [67,68]. In order to solve these problems, alginate biomaterials (surfaces) were modified with cationic polymers (that have the N-H groups). The alginate (-COO-) and cationic polymers’ functional group’s interaction, improve the degradation, cell adhesion, cell proliferation and the cell spreading of properties of the hydrogels were achieved. Therefore, in order to enhance the alginate applicability in extrusion 3D bioprinting, it should combine with other polymers to improve the bio ink properties (Supplementary Table II) and the 3D hydrogel characteristics. In a versatile method to create a 3D hydrogel, alginate is often combined with gelatin (well-known biomaterial ink composition), which extrudes into bioprinting [69]. The biomaterial ink developed can enhance the cell compatibility, printability, durability (prolonged use), biological activity and hydrogels’ stiffness [69]. In addition, the high stiffness of hydrogels can enhance the differentiation of the cells. Subsequently, the addition of chitosan (1.996 g) improves the viscosity of alginate (2 g) ink by between 1.5 and 4 times and also the shape fidelity (due to the electrostatic interaction) of the hydrogels [70]. The change in pH (by adding HCℓ) and the chitosan amine groups can alter the ammonium groups in the acidic medium of biomaterial ink. This composition can perform a crucial role in improving the biomaterial ink and the 3D printed alginate-chitosan polyion complex hydrogel properties. An optimized ink was employed to print the nose by using a 3D-BioplotterTm extruded printer (a 400 μm nozzle, N₂ gas). The 3D printed hydrogels that resulted, are biocompatible with human adipose-derived stem cells and they are highly useful for human organ development in tissue engineering applications.

The alginate molecule does not promote cell adhesion and therefore, cell proliferation may be absent in the cell adhesion motifs. The cell adhesion and multiplication promotion can be achieved by modifying the alginate by employing other functionalization groups, e.g., peptide biomaterials, which can mimic tissue-specific structures and support the structural fidelity of 3D biomaterials. In 1999, alginate was functionalized with arginine-guanidine-aspartate (RGD) by a carbodiimide chemical reaction [71]. According to the reports, RGD functionalized alginate, highly improved the cell viability and adhesion of the hydrogels (Rowley & Mooney, 2002). Furthermore, the high RGD functionalized alginate (high G blocks) can enhance the cells’ seeded and proliferation rates on alginate hydrogels [72]. Recently, different ratios of RGD and tyrosine-isoleucine-glycine-arginine (YIGSR) peptides (1:0, 0:1, 1:2 M) were conjugated with alginate [73]. The resulting (2 w/v%) peptides conjugated alginates, the Schwann cells and the brain-derived neurotrophic factors were used to produce a biomaterial ink for 3D bioprinting (3D Bioplotter, EnvisionTEC, Germany) at different conditions. Overall, a 30 kPa extrusion pressure and an 18 mm/s extrusion speed were found suitable for designing the 3D alginate hydrogels (pore printability ⁓0:95–1.0) with 50 mm CaCℓ₂. The RGD/YIGSR conjugated alginate hydrogels exhibited an acceptable swelling (584 ± 15.06 up to 24 h) characteristic, good degradation profile (52.63 ± 3.689 after 21 days), suitable/desirable elastic modulus (70% lose within 2 weeks) properties and with an acceptable cells viability. Therefore, peptides conjugated alginates (polysaccharides) are recommended for long-term in-vivo studies in the biomedical nervous system (peripheral ner) applications.

Alginate can reduce metal toxicity and improve the biocidal activity (cancer and antimicrobial) of hybrid nanomaterials. The general mechanism of cell toxicity is the carboxylate groups of alginate hydrogel that induce the electrostatic interaction with the metal ions (bioactive compounds) released (Fig. 3A). This binding can be influenced by the dispersion of metal ions on the alginate hybrid surface matrix and increases the hydrogel surface area, thereby, enhancing the ROS generation. In our earlier report, sodium alginate-ZnO hybrid nanomaterial exhibited higher anticancer properties against HepG2 cell lines when compared to ZnO and chitosan-ZnO nanomaterial [74]. In addition, toxicity studies were carried out on the L929 fibroblast by using alginate-ZnO hybrid nanomaterials and they exhibited minimum toxicity when compared to the ZnO and chitosan-ZnO nanomaterials. In this study, the alginate molecule enhanced the anticancer properties (against HepG2 cell lines) and increased the biocompatibility (reducing the toxicity of ZnO NPs on L929 cells) of the nanomaterials. Therefore, these alginate-based nanomaterials do not provide toxicity to the cell lines in a bio ink. However, non-toxic nanomaterials need to be loaded into the bio ink in order to achieve effective biomaterials for in-vitro and in-vivo applications.

Fig. 3.

A) The biocidal mechanism of carboxylate groups of alginate hydrogels and B) Schematic diagram of the co-axial 3D bioprinting of core-shell fibers/scaffolds for localized cancer therapy. Drug release from the core hydrogels after NIR-induced reversible sol-gel transition. Reproduced with permission [78], Copyright 2020, Elsevier.

In another investigation, alginate-gelatin hydrogels-based scaffolds were proven to enhance photothermal activity and they are biocompatible and are biogenic materials for cancer treatment and tissue repair applications [75]. These materials have proved to exhibit excellent physical, chemical and biological (environmental) stimuli. However, temperature is an essential component in any cell development in-vitro experiments. Unless a specific research goal is required, mammalian cells are typically grown at 37 °C in an incubator, supplied with 5% CO₂. Gonzalez-Fernandez et al., recently reported that alginate-gelatin (alg-gel) bio ink was dissolved in a phosphate buffer saline (PBS) for 10 min at 37 °C in order to obtain an effective bio ink that did not harm the mesenchymal stromal cells [76]. The Alg-Gel bio inks used for 3D bioprinting and its arm were kept at 10 °C throughout the measurement process by using the rheometer’s liquid temperature-controlling components, according to Li et al. [77]. However, during the extrusion in a 3D bioprinting process, the extrusion time must be kept at a specified temperature (37 °C or below) in order to crosslink alginate with other components, without harming the cells. Overall, alginate is a biocompatible substance used to form biomaterials ink.

Dopamine is a monomer and it can be oxidized and self-polymerized as polydopamine and used for the modification of alginate biomaterial ink and hydrogel [75,78]. In addition, polydopamine can impart significant physical and chemical interactions with alginate [75]. Core-shell shaped construct-based hydrogel fiber scaffolds (using 3D bioprinting) combined with near-infrared irradiation (NIR), triggered the drug release of therapeutics for localized therapy of breast cancer (Fig. 3B) [78]. In order to achieve such a print core-shell, the hydrogels need to have two different types of biomaterial inks: alginate (15.3 wt %), polydopamine inks as the shell layer and doxorubicin hydrochloride-loaded gelatin (temperature-sensitive) ink as the core part. Then, the biomaterial ink was loaded in the extrusion-based 3D bioprinting with co-axis core/shell nozzles needed to print the required 3D scaffold hydrogels at a temperature of between 25 and 60 °C with a printing speed of 5 mm/s and a pressure of between 300 and 400 kPa. Polydopamine has a significant photothermal effect; it can raise the temperature of the hydrogels under NIR (controlled drug release). Therefore, it can improve the photothermal effects of the shell and drug release capacity of the core of the hydrogels. Overall, their investigation, principally in-vivo studies on rat modal and other biological experiments, explained the fact that the polydopamine/alginate fiber scaffolds are potential candidates for localized therapy to treat breast cancer.

Researchers have employed alginate to develop 3D biomaterials for advanced medical applications. Since alginate (carboxylic acid group) can easily cross-link (ionotropic gelation) with divalent cations, this can result in enhanced bioink (gelation and mechanical) characteristics for better printability. However, in the extrusion in a 3D printing process, the composition of ink, nozzle diameter and printing speed are also essential. Therefore, hydrogel inks from alginate and gelatin methacryloyl [66], were prepared in order to mimic cartilage mimetic composites. In order to achieve such composite hydrogels, multiple tool biofabrication techniques were employed to load bone marrow-derived stromal cells and chondrocytes in the 3D printed alginate/poly(ε-caprolactone)-based biomaterials, which can be helpful for articular cartilage applications [66]. Similarly, gelatin methacrylate, two different (in viscosity) alginate polymers and a 14–3-3ε protein were used to prepare suitable biomaterial inks in order to print hydrogels by using a BioScaffolder bioprinter (200 μm inner diameter nozzle, a pressure of between 60 and 80 kPa and a 10 mm/s speed). These hydrogels were ionic (0.1 M CaCℓ₂) and chemically cross-linked (at 365 nm 45 W UV-LED array, 4 m W/cm²) in order to avoid hydrogel structure collapse [10]. However, the pore size and the printing accuracy can be increased by increasing the alginate viscosity in the ink. An extrusion 3D bioprinting has been used to prepare hydrogel scaffolds with cells (without living cells) in tissue engineering applications [79]. All in all, multiple investigations have suggested that the alginate-based hydrogel scaffold’s porosity, promotes neural cells differentiation and patterning and that it is valuable for spinal cord repair [66].

Recent investigations have suggested that the hydrogel properties (rigid and elastic) can be controlled (or improved) by forming a double cross-linking network by employing different polymers and components. One example is a catechol functionalized ink system from catechol-modified hyaluronic acid, alginate and gelatin [80]. It was used as ink for a 3D coaxial printing (printed with core and shell injectable channels, GeSIM BioScaffolder, Germany) in order to print tough and elastic hydrogels under (pressure for a core/shell product at 80/15 kPa, speed 6 mm/min), under specific conditions at room temperature. However, alginate was ionically cross-linked (with CaCℓ₂), which improved the strength of the hydrogels produced. Furthermore, gelatin was conjugated with catechol-modified hyaluronic acid via the Michael’s addition procedure between the catechol of hyaluronic acid and the amine/thiol groups of the gelatin, thereby, improving the cell adhesion and migration. In addition, the catechol-modified hyaluronic acid, used for self-crosslinking (H₂O₂ and horseradish peroxidase), has been produced to form hydrogels.

Gelatin-cellulose-alginate-based biomaterial ink has been produced through extrusion via the 3D bioprinting process in order to print cytocompatible blend hydrogels [81]. These biopolymer-based biomaterials (such as collagen and gelatin), have good cell-adhesion properties, but they have poor printability and long crosslinking duration. In order to attain better properties of the biomaterial ink, the authors used alginate and cellulose microfibers in the ink. According to their findings, when alginate content was increased (5:1:2) in the biomaterial ink composition, it attained a good viscosity, however, it was not suitable for printing due to its lack of homogeneity. In addition, when the cellulose content was increased (5:2:1), it was too stiff and unsuitable for printing. However, 5 (gelatin):2 (cellulose):2 (alginate) blended ink ratio was suitable for extrusion in a 3D bioprinting process (nozzle diameter 0.7 mm and printing speed is 10 mm/s) and the printed materials are shown in Fig. 4. The composition reported had a shear thinning behaviour and printing fidelity, while the 5:0:2 (gelatin: cellulose: alginate) ratio ink had a shear thinning behaviour. The cross-linking of the hydrogels utilized glutaraldehyde to (covalently) cross-link with CaCℓ_2, which can ionically interact with polymers. They observed that glutaraldehyde could reduce the smoothness and give color to the printed materials. Therefore, they concluded that CaCℓ₂ was better for the post crosslinking of printed materials and they did not show any toxicity on the fibroblast cells and they concluded that the materials were valuable for wound dressing applications.

Fig. 4.

Comparison of 3D printed structures. A) A filled cylindrical structure of Gel-Cel-Alg blends with wt/vol percentages of i) 5:0:2 and ii) 5:2:2, iii) the side view of 3D scaffolds printed in the absence (left) and presence (right) of cellulose in the composition, B) a cone structure printed with 5:2:2 blend, C) a hollow cylinder structure with 22 layers, printed with 5:2:2 blend, D) a mesh structure with cylindrical holes printed with 5:2:2 blend: i) top view and ii) side view, E) a cubical mesh structure printed with 5:2:2 blend, and F) an artificial ear prototype printed with 5:2:2 blend. All structures were printed with 0.7 mm nozzle and 10 mm/s printing speed at room temperature and cured with 10% CaCl₂ solution. Reproduced with permission [81]. Copyright 2020, Wiley-VCH GmbH.

Alginate is also used to prepare bioengineered implant materials for healthcare applications. Cheng et al., used alginate as a rheological modifier to manufacture a 3D printed material that is a free form of architecture of chemically and physically cross-linked hydrogels by using the direct-ink-write printing process [82]. The hydrophilic, ionic cross-linking network of alginate could play a crucial role in enhancing the mechanical toughness of the 3D printed hydrogel (Fig. 5) for biomimetic soft robots. Fig. 5 explains the fact that the biomaterial inks have water-like fluidity, low viscosity (η), low storage modulus (G′) and shear yield stress. However, when alginate and calcium cations are induced in the composite, its rheological (shear thinning) behaviors changed (Fig. 5b) for direct-ink-write printing of sophisticated 3D hydrogels (Fig. 5c), which was formed by chemical bonding and physical entanglement. Fig. 5d explains the biomimetic soft robotic systems. Ultimately, alginate was used to modify the rheological properties of several polymer-based biomaterial inks, prepared directly and used to print a bioengineered robotic heart.

Fig. 5.

Schematic illustration of direct-ink-write 3D printing hydrogels into biomimetic soft robots, (a) hydrogel precursor solution photo (acrylamide as an example here) with the composition diagram and the typical water-like rheological behaviour, (b) printable direct-ink-write ink photo (acrylamide direct-ink-write ink as an example here) with the composition diagram and the typical gel-like rheological behaviour after rheological modification, (c) digital design and printing of direct-ink-write ink (schematic) and the composition diagram after hydrogel curing and (d) biomimetic soft robotic systems (schematic) including an artificial tentacle, a bioengineered robotic heart and an artificial tendril. Reproduced with permission [82]. Copyright 2019, American Chemical Society.

The development of antimicrobial inks and additives might reduce the risk of microbial infections, hence, determining the success of tissue regeneration in humans. The antibacterial properties of the alginate-based inks and their hydrogels, depend mainly, on the selection of suitable crosslinkers (Cu²⁺, Sr^2+, Zn²⁺, Ca²⁺, Ga³⁺, Ce³⁺, which can provide antibacterial activity) and other antibacterial components [84,85]. Rastin et al. reported that gallium (Ga³⁺)-crosslinked methylcellulose/alginate ink, exhibited a potent antibacterial activity towards Staphylococcus aureus and Pseudomonas aeruginosa bacteria, with a bactericidal rate of 99.99% [86]. In addition, the gallium crosslinked hydrogels developed had higher and better biocompatibility than the calcium-crosslinked ink and the ink had excellent printability. Therefore, they concluded that gallium cross-linked biomaterial ink is useful for skin regenerative medicine.

Alginate biomaterial ink was prepared with metal nanoparticles in order to print alginate composite for tissue engineering applications. A laser 3D printer, which has multiple modalities (drop-on-demand printing, microextrusion and fused filament fabrication) with an appropriate temperature to print alginate nanoparticle composite by using microextrusion (printer needle of 1.5 mm in diameter and 20 mm in length) under a pressure of 0.2 bar, was employed [87]. Such laser printing with alginate cross-linked with CaCℓ₂ and with gelatin, can form a solid hydrogel.

The antimicrobial alginate/bacterial-cellulose hydrogels were used for 3D printed materials by substituting copper nanomaterials via an in-situ process [88]. By varying the alginate concentration from 1 to 4 wt%, the alginate-bacterial cellulose with Cu nanoparticles-based hydrogels have been printed with excellent 3D printability by using an extrusion-based 3D plotter. However, the 3D printing structures displayed good antibacterial activity against E. coli and S. aureus with varied susceptibility upon varying the concentrations of copper salts and for the designing of antimicrobial 3D-printed materials for microbial infection to reduce human pathogens’ risk.

Karavasili et al. demonstrated the 3D printable ink alginate-methylcellulose hydrogels, embedded with a bioactive compound, such as: Manuka honey, Aloe vera and Eucalyptus oil [89]. The carbohydrate-based 3D ink development has shown excellent swelling properties. The 3D-printed hydrogels showed typical biocompatibility at the lowest 0.001 g/mL concentration and stimulated cell proliferation. Hydrogels formulated with bioactive components, often exhibit high antibacterial and antibiofilm activities (>80% inhibition) towards S. aureus and E. coli and hence, they can be employed for wound healing applications.

Multiscale porous scaffold originated from sodium alginate, collagen and enrofloxacin-loaded poly(ε-caprolactone) microspheres by extrusion via 3D bioprinting technique, has been reported [90]. This scaffold is an effective hydrogel ink, attained by sodium alginate, which was cross-linked with Ca²⁺ (hydroxyapatite) and collagen with genipin. This pre-double cross-linked composite hydrogel inks, were injected by using a polypropylene syringe with a 0.84 mm screw dispensing needle. The authors stated that the porous scaffolds prepared, had good mechanical properties (2557 kPa at 70% strain), with control degradation. In addition, these scaffolds are not only biocompatible (with bone mesenchymal stem cells), they also have long-term antibacterial activity against human pathogens (Escherichia coli and Staphylococcus aureus), which can extend their applications to bone tissue engineering.

In order to encapsulate cells into the bio ink, it requires effective sterilisation techniques and they should not negatively, affect the properties of the optimized biomaterials ink. However, UV (ineffective) and ethylene oxide (cancerous chemical residue, flammability, highly toxic)-based sterilisation methods, are not suitable for sterilizing inks for extrusion via the 3D bioprinting [91,92]. Lately, Lafente-Merchan specified that the short cycle autoclave process (compared to the long cycle autoclave, β- and γ-radiations) is the best sterilizing cell-free alginate-based inks [91]. The nanofibrillated cellulose-alginate (NC: Alg) and the nanofibrillated cellulose-alginate-hyaluronic acid (NC:Alg-HA) biomaterial inks developed, were used in the extrusion-based 3D bioprinter to print scaffolds (27 G conical nozzle, pressure of between 20 and 22 kPa and speed 4 mm/s, with an extrusion pressure of between 24 and 26 kPa of NC:Alg-HA) for tissue engineering. In addition, hyaluronic acid improves the NC:Alg ink viscosity (500 Pa·s at 0.1 shear rate) values when compared to the pure NC:Alg ink (400 Pa·s). However, it can minimize the degradation behaviour of the scaffolds and it is less biocompatible with L929 cells when compared to NC:Alg. However, HA has improved the rheological properties of the alginate ink, which is an excellent bio ink for the development of scaffolds for regenerative medicine of the cartilage tissue. Overall, alginate biomaterial/bio inks properties can be improved by using multicomponent materials to achieve advanced biomaterials for biomedical applications.

5.1. Alginate with oxidized polymers for formation of biomaterial ink

Alginate and alginate derivatives can form potent physical cross-linking and improve the mechanical properties of the biomaterial ink, which can enhance the physical, chemical, and biomedical properties of the 3D-printed biomaterials [93]. However, hydrogels deformation can also occur due to the physical cross-linking interaction. In general, suitable (low) physiological stability (dynamic state of living organisms) and (high) shape fidelity have been problematic for the 3D printable alginate-hydrogels. Alginate hydrogels have lower compressive strength, low modulus and low dry matter, which can be of advantage as biomaterial/bio ink in extrusion-based bioprinting. The ink is loaded (storage modules) in cartridges and then extruded via conical or cylindrical nozzles [94]. Since inks should be easily movable inside the needle, during hydrogel printing by extrusion 3D bioprinting technique, it has been proven that the shape fidelity after printing, by employing different molecular weights and concentrations of alginates with appropriate viscosity, can be easily achieved.

Nonetheless, obtaining a high shape fidelity by printing, is difficult due to the alginate’s high water absorption capacity. In order to solve this issue, alginate is combined with other polymers and components to achieve suitable ink for 3D applications. Lately, the use of cellulose nanofibrils, high molecular-weight carboxymethyl cellulose, oxidized bacterial cellulose, tempo-oxidized cellulose nanofibrils, to prepare alginate-based ink, has resulted in good shear thinning (ease of extrusion) behaviour, self-supporting, shape fidelity and has been found suitable for 3D extrusion bioprinting in order to print hydrogels (physically crosslinked) without toxic reagents [94,95]. An equal ratio of tempo-oxidized cellulose nanofibrils and alginate-based biomaterial ink (66% viscosity recovery than the neat alginate), has the best printable property with the highest fidelity. 3D printed scaffolds are promising materials for bone tissue engineering applications (Fig. 6A and B) [94]. In the case of oxidized polymers, they have the carboxylate groups (COOH-) and they are responsible for forming a potently cross-linked network with alginate via a green process. Besides, hydrogel stability (in phosphate-buffered saline), is improved by adding nanomaterials and it can perform a long-term activity of the protein. Therefore, this phenomenon is helpful for drug release, biomedical devices and tissue engineering. Leppiniemi et al. formulated a biomaterial ink from oxidized nanocellulose, alginate, glycerine and avidin protein [96]. The avidin protein employed in its formulation has the amino groups, which resulted in the dynamic covalent bonding with oxidized nano celluloses and improved the biomaterial properties. They concluded that the resulting hydrogels can absorb water in moist environments and it is highly valued for wound dressing applications.

Fig. 6.

A) Fabrication process for 3D printing materials from tempo-oxidized cellulose nanofibrils/alginate-based biomaterial ink hydrogels and B) bone-related scaffolds printed in different shapes and designs from the optimized alginate-based biomaterial ink hydrogel, reproduced with permission [94], Copyright 2018, America Chem. Society.

Similarly, in order to improve the alginate ink capacity, another approach uses bifunctional cellulose (carboxyl and aldehydes) nanofibers, prepared from TEMPO-oxidization and periodate oxidation process [97]. These functional groups can form a stable acetal bond with polyvinyl alcohol (PVA) hydroxyl groups. This grating of polymer, provides flexibility and softness to the 3D printed alginate scaffolds. This study reported that a 15% of bifunctional cellulose, 2.5 g PVA and 5 g of alginate could form the best ink and it can be used to print a 3D material by using extrusion with a metal nozzle (0.5 mm diameter) at a speed of 1000 mm·min⁻¹ at a 0.05 bar pressure. It was concluded that these 3D-printed hydrogels are helpful for bone tissue engineering. It is worthy of note that alginate and oxidized polymers can provide effective ink for 3D bioprinting applications. Therefore, the oxidized polymers can improve the alginate applicability in a 3D technology.

6. Oxidized alginate (alginate dialdehyde) for 3D bioprinting technology

Polysaccharides have poor physicochemical properties (poor water solubility and bioactivity and they are challenging to degrade). In addition, they have shown few side effects in medical applications, due to their structures [98]. Therefore, they are modified in order to improve their functionality and applicability for effective bioengineering and healthcare applications. Water-soluble and bioactive (lower molecular weight) alginate can be produced via chemical (hydrolysis, oxidative degradation) and enzymatic (enzymatic digestion) methods [99]. In the oxidative degradation method, a degradable reagent that can produce water (oxidation by-product) and hydrogen peroxide, has been employed to produce a bioactive oxidized alginate (Ring opened alginate, Fig. 7: III). Oxidized alginate (which has dialdehyde functional groups) was synthesized [100] from the oxidation of sodium alginate, which was first prepared in 1928. The oxidation process of alginate is higher (50–60%) in an ethanol-water solution than in the aqueous solution [101]. Such oxidized alginate has more reactive groups, which can enhance the cell interaction and biodegradability of hydrogels. Due to their low molecular weights, oxidized alginate-based hydrogels have lower mechanical (flexibility) properties and higher degradation rates (due to their reactive functional groups or their hydroxyl free radicals) than alginate hydrogels. However, hydrogels properties (mechanical, swelling and degradation), depend mainly, on the covalent cross-linking between the polymers and the oxidation degree of alginate (molecular weight). A high degree of oxidation of alginate leads to the high density of the hydrogels (biomaterial ink) and hence, improves the drug release rate. Nevertheless, a lower density of the hydrogels, allows the cells to grow when compared to the high-density hydrogels. However, oxidized alginate (1–5%) can form low physical cross-linking with divalent ions (Ca²⁺) due to the lack of the α-L-guluronic blocks [102]. The elastic modulus and the slight biocompatibility of these gels decrease with an increasing degree of oxidation of the alginate. 9% oxidized alginate-based hydrogels are not harmful (cytocompatibility) to many living cells [102]. However, biodegradability is needed for temporary medical implants (made from natural polymers, with molecular weights below 50 kDa) after new tissue (healing wounds) formation and they should be removable from the body without any side effects [103]. Hydrogel’s degradation is controlled by the varying alginate molecular weight, chemical structure and covalent crosslinking. Nevertheless, this hydrogel’s degradation (Supplementary Table IV) is uncontrollable in different phosphate-buffered saline [104]. Additionally, alginate (high molecular weight) is very difficult to remove from the body because it cannot be enzymatically (naturally) degraded in mammals and it can take several years to remove it altogether [105]. Therefore, alginate is chemically modified as oxidized alginate in order to improve its biological properties.

Fig. 7.

I) Cross-linked hydrogels from unmodified and oxidized alginates [106] (Open Access This article is licensed under a Creative Commons Attribution License, MDPI), II) schematic of (A) static polymer chains with covalently cross-linked networks and (B) dynamic polymer chains with flexible, dynamic networks. Reproduced with permission [36] Copyright 2019, American Chemical Society. III) Schematic diagram of the formulation of alginate-based biomaterial ink (ADA-GEL) and IV) Printability assessment. A) Resolution structures. From left to right: CAD-File, ADA-GEL-T70 °C_6 h, ADA-GEL-T80 °C_3 h, ADA-GEL-T80 °C_6 h, ADA-GEL-T95 °C_3 h, ADA-GEL-TRef. B: 3D printed ADA-GEL-Grids. From left to right: ADA-GEL-T70 °C_6 h, ADA-GEL-T80 °C_3 h, ADA-GEL-T80 °C_6 h, ADA-GEL-T95 °C_3 h, ADA-GEL-TRef, Scale: 2000 μm. C: 3D printed ADA-GEL-Single-Layer. From left to right: ADA-GEL-T70 °C_6 h, ADA-GEL-T80 °C_3 h, ADA-GEL-T80 °C_6 h, ADA-GEL-T95 °C_3 h, ADA-GEL-TRef, Scale: 2000 μm. D: 3D printed ADA-GEL-Strands. From left to right: ADA-GEL-T70 °C_6 h, ADA-GEL-T80 °C_3 h, ADA-GEL-T80 °C_6 h, ADA-GEL-T95 °C_3 h, ADA-GEL-TRef, Scale: 500 μm. E: Obtainable height stability using ADA-GEL-TRef. F: Obtainable height stability using ADA-GEL-T80 °C_3 h [111] (This is an open-access article and licensed under a Creative Commons CC-BY license, Elsevier).

The oxidation of alginate controls the degradation of hydrogels at different pHs and temperatures [106,107]. In addition, ionically (Ca²⁺) and enzymatically (microbial transglutaminase) cross-linked oxidized alginate/gelatin hydrogels, have good elasticity or stiffness (between 5 kPa to 120 kPa Young’s modulus) and good degradation behaviour (from 7 to 30 days) for tissue engineering applications [108]. These hydrogels showed desirable stress relaxation properties. However, ionically crosslinked alginate, often controls the biomaterials stress relaxation character.

Principally, oxidized alginate has negative charges and it can form a dynamic covalent bond with a free amine group of the biopolymers (chitosan, collagen and gelatin) or imine type cross-linkers and other materials (Fig. 7I). It has proved its self-healing (which can be produced via non-covalent interactions) and self-recovery (re-joining at the molecular level) capabilities to the hydrogels and 3D bioprinted materials [36,109]. According to the literature, dynamic covalent bonds (cross-links) are reversible in the polymer network (Fig. 7II) and such networks exhibit dual stimuli-responsive behaviour for temperature and shear response. However, oxidized alginate provides good ink, demonstrating very potent viscoelasticity, shear-thinning and sealing properties. Thus, the combination of biopolymers can improve the cell-binding (cell signaling) and proliferation capacities of alginate-based hydrogels in tissue engineering applications. However, according to reports, good biomaterial ink has a viscosity in the range of between 400 and 3000 mPa·s, which is suitable for cell loading and can be used for extrusion in a 3D bioprinting [110]. Kewller et al. reported an effective biomaterial ink from oxidized alginate (3.75% w/v) with gelatin (7.5% w/v) (ADA-GEL), which can provide a potent shape stability in order to obtain a better structure at the height of over 1 cm by 3D bioprinting (Fig. 7III and IV) [111]. Furthermore, this formulation has favourable (recovery) printability at 80 °C for 3 h when compared to alginate-gelatin-based biomaterial ink [111]. Overall, the biomaterial ink hydrogels degradation is controlled by using oxidized alginate and hence, an oxidized alginate-based ink is beneficial for the designing of complex structures and 3D bioprinted hierarchical templates for possible future biomedical applications [42].

7. Other alginate modifications

These modifications are towards the efforts to improve the alginate’s biological and functional activities that can be chemically modified via sulfation, phosphorylation, esterification and amidation. The alginate-sulfate is prepared via sulfation [112]. The alginate-sulfate can interact with several growth factors and it is used in the preparation of hydrogels to improve cell viability when compared to neat alginate [113]. Thus, alginate-sulfate is used in the formulation of bio ink in order to improve the growth factors delivery, cellular interaction and enhance the 3D printing processibility [114]. However, it had a limited influence on the rheological characteristics of the bio ink [115].

The phosphorylation of alginate is obtained by employing urea and phosphate or phosphoric acid reaction [116,117] and according to the reports, its hydrogels have better and suitable physicochemical and mechanical properties for in-vivo applications than the pure alginate hydrogels [116–118].

The esterification reaction is used to modify native alginate, which can increase the hydrophobic nature of alginate. In this reaction, alginate can be modified via its treatment with preferred alcohols in the presence of an appropriate catalyst. In addition, researchers have prepared a water-soluble modified alginate (amphiphilic) and another form of the alginate. However, the amidation process improves the alginate stability and bioactivity by increasing its hydrophobicity [116,119]. It is noteworthy to state that due to the modified alginate, practical functionality can result to the formulation of effective biomaterials/bio ink for advanced applications [120,121].

8. Nanomaterials influence on alginate biomaterial ink

This section discusses how nanomaterials can enhance the alginate-based biomaterial ink for the printing of 3D hydrogel structures (Table 1 and Fig. 8). Table 1 exemplifies the different nanomaterials that can be employed, highlighting their advantages. They also highlight the printing conditions of the various bio inks, the types of hydrogels that can be produced and their advantages, in addition to their study routes/techniques (in-vitro and/or in-vivo). Most importantly, the applications of these materials are highlighted. On the other hand, Fig. 8 highlights, schematically, the methods of production and sketches of the 3D printed materials.

Table 1.

Nanomaterials signification in the formation of alginate-based biomaterial ink and their biomedical applications.

Nanomaterials	Advantages	Printing conditions for ink	Hydrogels and their advantage	In-vitro/in-vivo studies	Applications
Layered rare-earth hydroxide (Europium, Terbium ions)	-Fluorescence characteristic -Used to Print at room temperature	Printing speed 2 mm/s, pressure 0.2 bar, a conical nozzle 400 μm	Multifunctional hydrogels which exhibit humidity dependent electromechanical properties Transparency, extensibility, tunable stiffness and highly tunable fluorescence (Fig. 8A), and sinlike sensor	In-vitro	Electronic skins [122].
Graphene Oxide	-Improve shape fidelity and resolution -Improve the cell proliferation -faster viscosity recovery during extrusion.	Micro-extrusion process: 25 G needle	Hydrogels (Fig. 8B) have high cell proliferation and chondroinductive (human adipose tissue-derived mesenchymal stem cells) due to bioconjugation	In-vitro	Cartilage tissue engineering [123].
Graphene oxide (2D carbon)	Improve mechanical performance -Graphene Oxide can interact with non-covalent and ionic crosslinking. - used for light directed 3D printing	Stereolithographic 3D printing with a 355 nm 60 m Wdiode-pumped solid-state UV laser	Hydrogels are ionically tunable and chemomechanical stable. Hydrogel covalent interactions (Fig. 8C) enable dynamic biologically inspired functionalities in marine environments.	In-vitro	Oil droplet manipulation and antifouling application [124].
Cellulose nanofibrils	-Enhance the printability of ink and provide a good shape fidelity.	Extrusion-based printing: printing pressure range 18–21 kPa, the diameter of the conical nozzle 410 μm.	Hydrogel has double networks (Fig. 8D) with good mechanical properties. They have high cell (human auricular chondrocytes) viability (more than 90%).	In-vitro	Carilage Engineering [125].
Copper and bacterial cellulose nanofibrils	Copper is used as an ionic crosser, antibacterial agent and improves printability. Bacterial cellulose nanofibrils can improve printability -Pintable at 25 °C	Extrusion pressure of 1 bar, printing speed 50 mm/s, 23 G needle tip	Antimicrobial 3D printed hydrogels (Fig. 8E). It can control Escherichia coli and Staphylococcus effectively	In-vitro	Tissue engineering and regenerative medicine [88].
SiO2	-Chemically interacts with polymers. -Improve the viscosity of ink and prove biocompatibility and osteogenic ability -Bone graft capacity	Extrusion pressure 35 Psi, printing speed 10 mm/s	Printed material compressive modulus significantly improved. However, its swelling, degradation characteristic is inhibited substantially. Hydrogel composite has good biocompatibility (human umbilical cord mesenchymal stem cells) and osteogenic capacity enhanced with SiO2 nanoparticles (Fig. 8F).	In-vivo	Bone tissue engineering and nanomedicine [126].
Laponite (nanosilicate clay)	-Improves printability, shape fidelity, and control the release of biomolecules	Extrusion: biomaterial ink extruded via a γ-irradiated conical plastic needle, it diameter 410 μm, pressure 80–90 kPa, speed 8–10 mm/s	Hydrogel (Fig. 8G) is used as a drug delivery system with high human mesenchymal stem cell viability.	In-vitro	Skeletal applications [127].
Laponite	-6% if laponite can easily extrude and improve the selfsupporting scaffold material -used for direct printing	Extrusion: 25 gauge (250 μm diameter), Pressure 1.52 × 105−1.72 × 105 Pa speed 1.0–0.75 mm/s	Printed hydrogels (Fig. 8H) have cytocompatibility (fibroblasts) and degradation resistance.	In-vitro	Biomedical (Direct-write) applications [128].
Hydroxyapatite	-Pre crosslink capacity and improve the bioactivity -Anti-inflammatory curcumin-loaded SiO2.	Biomaterial ink injected by using polypropylene syringe, needle diameter 0.84 mm	Porous hydrogels (Fig. 8I) have superior biocompatibility with mouse bone mesenchymal stem cells. Useful as a drug devilry system	In-vitro	Bone tissue engineering [129].
Magnetic nanoparticles	-Enhance the controllability	Microfluidic Printing: height of the microchannel is 100 μm, and the glass cover has 50 mm, width 10 mm, and thickness 0.12–0.17.	Magnetic alginate microfibers (Fig. 8J). Significant fibroblast cells viability.	In-vivo	Tissue engineering (in vivo tissue) [130].
Luminescent nanoparticles	-(O2 sensor) optical sensor -(2.5 mg mL−1) does not affect cell and improve the viability -Excellent printing fidelity and biocompatibility for printing hydrogels,	Extrusion pressure 80 and 300 kPa, conical needle 610 μm, speed 10 mm/s.	Cross-layered, wood-pile structured hydrogels biocompatibility with human cell lines (Fig. 8K).	In-vivo/in-vitro	Biomedical applications. (Sensor-functionalized ink has a wide range of applications) [131]

Fig. 8.

A) Skin-inspired multifunctional luminescent hydrogel. Reproduced with permission [57] Copyright 2020, American Chemical Society, B) chondroinductive alginate-based hydrogels having graphene oxide. Reproduced with permission [58] Copyright 2020, American Chemical Society, C) alginate-graphene oxide hydrogels. Reproduced with permission [59] Copyright 2018 Elsevier, D) double-network hydrogels including enzymatically crosslinked poly-(2-alkyl-2-oxazoline)s. Reproduced with permission [60] Copyright 2019, American Chemical Society, E) antimicrobial alginate/bacterial-cellulose composite hydrogels by incorporating copper nanostructures. Reproduced with permission [61] Copyright 2019, American Chemical Society and F) SiO₂ nanoparticles are loaded into alginate–gelatin composite hydrogels. This Open article is licensed under a Creative commons Attribution 3.0 Unported License [62]. G) Nanocomposite from laponite, alginate, and methylcellulose. Reproduced with permission [63] Copyright 2017, IOP Publishing H) self-supporting direct hydrogel, which has self-supporting nanoclay. Reproduced with permission [64] Copyright 2017, American Chemical Society, I), sodium alginate/hydroxyapatite hydrogels. Reproduced with permission [65] Copyright 2019, John Wiley and Sons, J) magnetic assembly of microfluidic spun alginate microfibers. Reproduced with permission [66] Copyright 2015, Springer Verlag Berlin Heidelberg and K) Optical Sensor Nanoparticles for O₂ Imaging in 3D-Bioprinted Constructs. Reproduced with permission [67] Copyright 2018, John Wiley and Sons.

Alginate has been used to prepare biomaterial/bio ink (additive) due to its biocompatibility and printability. However, several studies have reported that alginate has limited rheological properties and that these properties were improved by adding viscous polymers (modified polymers) and nanomaterials [132]. Mainly, nanomaterials improve the printability and mechanical strength of alginate-based biomaterial ink [83]. For example, a 2% inclusion of nanosilicates, not only improves the (alginate-gelatin-rat bone marrow mesenchymal stem cells) biomaterial ink properties, it also heals the bone without any side effects on the recipients due to its osteoinductive character [83]. Furthermore, this biomaterial ink-printed scaffold, supports rat bone marrow mesenchymal stem cell viability (Fig. 9) and heals the rat bone defect (critical size 8 mm) within 12 weeks, indicating the fact that they are successful in orthopaedic applications. Another biomaterial ink, composed of graphene oxide (0.5 mg mL⁻¹) and 3% of alginate, can offer easy printability, improve the structural stability, osteogenic and cell viability of the hydrogel (human mesenchymal stem cells) [133]. The biomaterial ink was used to print the composite at a feed rate of 25 mm/min and a pressure of 87 kPa by using a 22G nozzle. According to another study, nano-hydroxyapatite (1%wt) promotes osteogenesis in the alginate (2 wt%), gelatin (8 wt%) and human adipose-derived stem cells-based biomaterial ink and 3D printed hydrogels [134]. This biomaterial ink hydrogel has high cell viability, adhesion and proliferation. These observations were recorded by using an immunofluorescence microscope.

Fig. 9.

The laponite nanocomposite-alginate-based biomaterial ink supports cell viability. A. Schematic of the 3D bioprinting process using a 2% of nanosilicates biomaterial ink. B. Live/dead cell staining of the 3D bioprinted scaffolds. 3D reconstruction of the confocal microscope images showed numerous live cells (stained green) that were uniformly distributed within the 3D bioprinted 2% nanosilicates scaffolds after 1, 3, 5, 7, 14 and 21 days of culture in a normal medium in-vitro. In addition, Laponite nanoparticles were stained red due to electrostatic interactions with ethidium homodimer-1 [135]. Scale bar: 100 μm. C. Hematoxylin and eosin stain (HE stain) of sections of the bioprinted scaffolds (2% nanosilicates) after 7, 14 & 21 days of culture in normal medium. Scale bar: left column: 200 μm, right column: 50 μm. Reproduced with permission [83]. Copyright 2020, Elsevier.

The polyacrylic acid-magnetic nanoparticles and methylcellulose improve the rheological behaviour of the alginate-based biomaterial ink [132]. The biomaterial ink that was composed of alginate, viz: methylcellulose = 1:1 + polyacrylic acid: magnetic nanoparticles = 1:2, exhibited favourable shear-thinning behaviour, which meant that the viscosity of the biomaterial ink was reduced with the increasing share rate. Thus, this thixotropic (shear stress versus shear rate plots) ink can be used directly for magnetic hydrogel and their 3D-printed objects could remotely, be actuated via magnetic fields. Another construct, based on carboxylated-cellulose nanocrystals and alginate/xanthan gum-based hydrogel, has improved the shear-thinning behaviour of hydrogel ink and its printability of the hydrogels [136]. The hydrogel-ink developed was used to print hydrogel by direct-write extrusion-based 3D printing (at 25 °C, speed 1.0 mm/s, pressure of between 45 kPa to 95 kPa, 25 gauge, which has an inner diameter of 410 μm). However, the authors specified that a 0.36 g of carboxylated-cellulose nanocrystals-based hydrogel (alginate 0.63 g, 0.0375 g and water 25 g) ink, had a potent printing stability and shape fidelity. In addition, it improved the mechanical and viability properties of human skin fibroblast cells. They concluded that their continued efforts would be devoted to achieving an effective 3D hydrogel biomaterial ink for tissue engineering applications.

100 ppm silver nanoparticles (10 mg mL⁻¹, which is the minimum inhibitory concentration) was incorporated in an alginate/nanocrystalline cellulose (5%/3% w/v)-based biomaterial ink 3D printing scaffolds, which significantly promoted antimicrobial and cytotoxic effects and thus, made it a valuable product for wound dressing applications [137]. An egg-box shaped scaffold structure was achieved for this biomaterial ink hydrogel when it was cooled at −14 °C and it had a viscosity of between 8 and 40 k cP after printing and crosslinked with 3 M CaCℓ₂ for 30 mints. However, the pure alginate hydrogel developed, dispersed faster due to its low mechanical properties than the alginate/nanocellulose composite, which has high mechanical stability and is useful for clinical applications.

Bergonzi et al. did demonstrate that 3D printed nanocrystalline cellulose in the alginate/Ag hydrogels, improved the scaffold’s mechanical properties and cytotoxicity against hepatocellular carcinoma (HEPG2) cells, which is more effective over time [137]. In addition, Guduric et al. have reported that Zn-containing mesoporous bioactive glasses, coated with alginate-methylcellulose blend, exhibited desirable rheological properties that are suitable for 3D printing of scaffolds and that they have sufficient shape fidelity, which results in a tailorable bioprintable material system that is ideal for bone tissue engineering and antimicrobial applications [138].

For the extrusion-based 3D printing procedure, it depends mainly, on the supramolecular biomaterial ink properties [139]. The suitable biomaterial ink has low viscosity, however, it can permit the extrusion process and it can undergo a rapid sol-gel transition in order to obtain the appropriate hydrogels. Therefore, it can be modified via chemical crosslinking, photo-crosslinking and ionic crosslinking in order to obtain a rapid sol-gel change to stable 3D printed hydrogels without losing their mechanical properties, a photo-cross-linkable biomaterial ink that was developed from alginate, nano clay (Cloisite^® 20A) and acrylic acid solution [139]. Electron beam irradiation (5–60 kGy) produced free radicals and reacted with alginate, clay and acrylic acid to form a cross-linking network structure. It was used for the extrusion-based printing to obtain hydrogels (needle diameter 1 mm, speed 15 mm/s, the flow rate of ink 0.40 mL min⁻¹) at ambient temperature, without any postprint cross-linkering process. The printed nanocomposite hydrogels were treated with radiation of between 15 and 45 kGy. However, lower irradiation-treated 3D printed hydrogel, has a better adsorption capacity than other hydrogels. Overall, these hydrogels quickly adsorb heavy metals within a short time and are helpful in wastewater treatment and other biomedical applications [139]. Zhang et al. successfully launched a biomaterial ink formulation from spherical colloidal lignin particles (CLP), cellulose nanofibril (CNF) and alginate [140]. It is worth mentioning that the CLP (0.05 wt%) did not modify the shear-thinning behaviour, but it improved the printing resolution. CLP provides antioxidant properties and improves the shape fidelity of the printed hydrogels with a high swelling capacity and biocompatibility with the hepatocellular carcinoma cell line. In another strategy, polylactic acid nanofibers incorporated in alginate hydrogel biomaterial ink, improved the cell viability of human adipose-derived stem cells [141]. In general, nanomaterials can enhance the alginate-based biomaterials ink properties for practical 3D printing applications. Therefore, biologically active biomaterials ink, extends the possibility to design the next generation of biomaterials for healthcare applications.

9. Future scope and challenges

Organ development is growing with the use of 3D bioprinting technique, with the intention of helping the lack of donated organs and to stop illegal organ trading on the black market [142,143]. Testing new drugs on printed tissues (kidneys and liver) without burdening or exposing the patient’s organs to side effects is an added benefit. The 4D printing technology uses programmable and advanced materials, takes it further with the creation of 3D biological parts with shapes that respond to time-dependent functions. Indeed, time is the extra 1D dimension and the shape change is caused by issues/concerns, such as: water, temperature, or light. However, multifunctional/multi-component biomaterial/bio ink hydrogels can be synthesized from organic, inorganic and other bioactive materials and activated by light irradiation [144]. Biomaterial/bio ink hydrogels must meet specific health and safety standards when they are made. These include: low cost, small size, high yield, tunable properties, biocompatibility, multi-functionality and controllability. As a result, researchers can create long-lasting and high-performing dental and other body prostheses without worrying about toxicity or side effects. However, in future, effectively modified polymers with multifunctional/multi-component biomaterial ink hydrogels are expected to achieve self-healing potency and self-recovery biomaterials that can be used in human healthcare applications. In addition, practical, simple process handling and economically feasible 3D technology and 3D printed materials are to be further desired, for advanced human and animal healthcare applications.

10. Conclusion

In the 3D bioprinting technology, biomaterial/bio ink hydrogel formation affords an active platform for the design of the next generation of innovative 3D-printed biomaterials for biomedical applications. However, as mentioned, alginate has poor bioactivity, however, it can be improved by modifying alginate’s chemical (molecular weight) structure. In addition, alginate ink has poor physiological stability, shear-thinning behaviour and shape fidelity, which can be enhanced by adding viscous modifying polymers and nanomaterials in order to obtain advanced biomaterials/bio ink for bio-fabrication of suitable biomaterials. This review article has focused on the considerable information that are available on recent investigations on alginate, oxidized alginate and the various nanoparticles of significance that are needed to produce alginate biomaterial/bio ink for 3D printing applications (with printing conditions). Principally, alginate and its derivative (oxidized alginate) biopolymers are nontoxic, biocompatible, biodegradable, biostable, biogenic and they significantly, influence the rheological properties (viscoelasticity, shear-thinning and sealing properties) of biomaterial ink. Thus, they can, obviously, advance the cell-binding and proliferation capacities of alginate-based 3D printed hydrogels for their future use in biomedical applications. Additionally, alginate’s physical and chemical interactions with several polymers (with N-H functionality) can improve the cell-binding, swelling and degradation tendencies of 3D printed hydrogels. In addition, nanomaterials can improve the shape fidelity and the resolution of 3D printed scaffolds. Overall, alginate-based biomaterial/bio inks have been used in the construction of the best biomaterials for different biological/biomedical applications.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijbiomac.2022.05.157.