**2. Application of alginate**

The requirement for alginate-based biomaterials in drug delivery and tissue engineering is huge. As stem cells play a progressively more major function in the area of regenerative medicine [14, 15], the arrangement and relation between alginate-based materials and stem cells have been exclusively emphasized. Investigated by in vitro implantation and in vitro cytotoxicity assay, alginate-based scaffolds and microcapsules have shown minimum or minor cytotoxicity [16, 17]. These in vitro results recommended tunable connections between the bio-composites and the multiple platelet releasate-derived bioagents for improving hematoma-like fracture repair. Also, a simple invasive performance for in situ remedial of the implant structures through injection was established in rat tail vertebrae applying microcomputed tomography. These results confirmed that alginate-based scaffolds

were capable of degrading, permitted the vascularization, and obtained minimum inflammatory responses after transplantation. Consequently, alginate-based scaffolds can present suitable characteristics as probable cell and drug carriers for tissue regeneration. The next sections explain the clinical and preclinical analysis of alginate-based biomaterials and applications.

#### **2.1 3D bioprinting**

Sodium alginate which is also known as sodium alginate or algin is a naturally extracted less costly polymer from the brown algae cell walls which have intracellular spaces [10]. Alginate is composed of (1-4)-linked β-d-mannuronic (M) and α-l-guluronic acids (G). Alginate is a polyanionic linear block copolymer made of longer M or G blocks, separated by MG regions. Sodium alginate is a kind of polysaccharide which is charged negatively because it is known that materials which are positively charged produce inflammatory response; this allows the biopolymer to support high biocompatibility and cell growth. G blocks enhance the gel structure and M and MG blocks enhance the elasticity, though a high quantity of M blocks could be the reason of immunogenicity [18]. In alginate matrix, with the help of capillary forces, water and other molecules can be trapped. This feature makes alginate hydrogel suitable for bioink designs. The cell density within the bioink will be very high, whereas the shear stress through the extrusion process decreases the cell viability (80–90%). In the case of inkjet bioprinting, the bioinks have lower cell densities (<16 × 106 cells/mL) and are less viscous (<10 mPa s). This technique suggests 90% cell viabilities but, in laser-assisted bioprinting, needs bioinks with viscosities of 1 and 300 mPa s and also requires medium cell densities (108 cells/ mL). In this method, cell viability is very high (>95%). The alginate-based bioink viscosity rests on the alginate molecular weight, alginate concentration, density of cells, and cell phenotype. These are the variables that scientist must consider to optimize the viscosity of the alginate-based bioinks. An additional significant rheological characteristic of aqueous alginate solutions is the shear-thinning, where the shear rate increases while decreasing the viscosity. The viscosity also depends on the performed printing temperature; when the temperature increases, the viscosity gradually decreases. In comparison with other polymers, alginate is convincingly easy to handle and to print and is easy to extrude (printing) while defending the encapsulated cells. Even if it is also a non-cell-adhesive [12], in case of cell encapsulation, alginate is currently one of the most applied biomaterials. After the printing performance, the hydrogel should degrade suitably, allowing the cells to make their specific extra cellular matrix (ECM). The alginate also generates durable insistent cell-laden hydrogels; however oxidation can be performed by slow degradation, for example, sodium peroxide [11]. The main issue of alginate for using it as a biomaterial in bioprinting is slow degradation rate. The release of the hydrogels through the bioprinter nozzle in bioprinting (extrusion) limits the usage to low weight of alginate, which has a major role in the application of reduced mechanical properties. Though the alginate mechanical and structural characteristics are needed for all printed tissue, the biomimicry characteristics required in every instance can be changed by combining new biomaterials in the scaffold or by applying different types of hydrogel fabrication technique. For example, CELLINK is a commercial bioink which is already available for bioprinting; it combines with alginate hydrogel and nanocellulose and presents fast cross-linking and shear-thinning properties; this bioink is appreciated for soft tissue engineering for bioprinting [8]. The formation of blood vessel-like channels is able to transport different materials like nutrients and oxygen via the bioprinted material, which is needed in order to print organs or tissues. To succeed in this aim, Zhang et al. [19] made vessel-like printable

**113**

added cells is shown.

**2.2 Wound healing**

*Importance of Alginate Bioink for 3D Bioprinting in Tissue Engineering and Regenerative…*

showed the controlled degradation of oxidized alginate in 3D bioprinting.

Varying biodegradability of solution of sodium alginate along with human adipose stem cells was printed with accurate definition. These kinds of bioinks have the capacity to modulate proliferation and stem cell spreading and withstand uniform cell suspension but are imperfect in the case of stem cell diffusion. Wu et al. [25] showed the procedure of slow degradation of the alginate by tissue incubation in a sodium citrate medium. The sodium citrate amount helped the optimization of the alginate degradation time. Chung et al. [18] improved the printing resolution and printability of pre-crosslinked printed constructs by adding alginate with gelatin, keeping the mechanical property and the growth of cells, and keeping pore diameter constant. In **Figure 3** the procedure of alginate cross-linking with the

Alginate has been used for dressing of the wounds due to its of good conformability, absorptivity, and mild antiseptic properties coupled with biodegradability and nontoxicity and optimal water vapor transmission rate. Alginate-based products like electrospun mat hydrogels and sponges in dressing of wounds are very good substrates for healing of wounds, which include gel-foaming capability as soon as the absorption of the wound exudates and hemostatic capabilities [26]. It is already been mentioned that dressing wounds with alginate improves healing of wounds through monocyte stimulation to harvest higher cytokine levels like tumor necrosis factor-α and interleukin-6 [27]. Near the wound locations, cytokine production creates pro-inflammatory factors that are helpful for wound healing. Because of the existence of endotoxin in the alginate, a huge level of bioactivity

microfluidic channels where a coaxial nozzle strategy is used for transporting the nutrient into printed material, and the printer was pressure-assisted bioprinter and the coaxial needle was applied for printing the hollow alginate filaments that contain cartilage progenitor cells. In the same way, a triaxial nozzle assembly was used to fabricate biocompatible cartilage-like tissues containing tubular channels, where the alginate was encapsulated by cartilage progenitor cells, which is the main element of the bioink. Hydrogels of sodium alginate having high strength and having inner microchannels were found out by Gao et al. [20]. Also, constructs like perfusable vascular-like constructs were also obtained through coaxial multilayered nozzle along with the concentric extrusion channel by 3D printing in one step [21] by mixing 4-arm poly(ethylene glycol)-tetra-acrylate (PEGTA) and gelatin methacryloyl (GelMA) with the sodium alginate. Calcium ions were used to cross-link the alginate, and photo-cross-linking was used for covalent cross-linking for PEGTA and GelMA for setting the rheological and mechanical properties that was reported in this work. Also in another study, Christensen et al. [12] printed vascular structures along with bifurcations (vertical and horizontal) in alginate and fibroblast of mouse bioinks. Blending alginate with other polymers (honey, gelatin) [22], amino acids like polyglutamic acid and poly-l-lysine [13, 23], and some drugs like N-acetylcysteine (NAC) [24] was studied for improving the erratic degradation, cell-material interaction, cell viability, etc. The printer (inkjet) used CaCl2 cross-linking agent supporting material for cross-linking the alginate bioink. To back up the buoyant force in the regions overhanging in both vertical and horizontal printing and also supporting the regions spanning in the horizontal printing, their modified solution was used. Blending alginate with other polymers (honey, gelatin) [22], amino acids like (polyglutamic acid and poly-l-lysine) [13, 23], and some drugs like N-acetylcysteine (NAC) [24] was studied for improving the erratic degradation, cell-material interaction, cell viability, etc. Jia et al. [6] in their study

*DOI: http://dx.doi.org/10.5772/intechopen.90426*

#### *Importance of Alginate Bioink for 3D Bioprinting in Tissue Engineering and Regenerative… DOI: http://dx.doi.org/10.5772/intechopen.90426*

microfluidic channels where a coaxial nozzle strategy is used for transporting the nutrient into printed material, and the printer was pressure-assisted bioprinter and the coaxial needle was applied for printing the hollow alginate filaments that contain cartilage progenitor cells. In the same way, a triaxial nozzle assembly was used to fabricate biocompatible cartilage-like tissues containing tubular channels, where the alginate was encapsulated by cartilage progenitor cells, which is the main element of the bioink. Hydrogels of sodium alginate having high strength and having inner microchannels were found out by Gao et al. [20]. Also, constructs like perfusable vascular-like constructs were also obtained through coaxial multilayered nozzle along with the concentric extrusion channel by 3D printing in one step [21] by mixing 4-arm poly(ethylene glycol)-tetra-acrylate (PEGTA) and gelatin methacryloyl (GelMA) with the sodium alginate. Calcium ions were used to cross-link the alginate, and photo-cross-linking was used for covalent cross-linking for PEGTA and GelMA for setting the rheological and mechanical properties that was reported in this work. Also in another study, Christensen et al. [12] printed vascular structures along with bifurcations (vertical and horizontal) in alginate and fibroblast of mouse bioinks. Blending alginate with other polymers (honey, gelatin) [22], amino acids like polyglutamic acid and poly-l-lysine [13, 23], and some drugs like N-acetylcysteine (NAC) [24] was studied for improving the erratic degradation, cell-material interaction, cell viability, etc. The printer (inkjet) used CaCl2 cross-linking agent supporting material for cross-linking the alginate bioink. To back up the buoyant force in the regions overhanging in both vertical and horizontal printing and also supporting the regions spanning in the horizontal printing, their modified solution was used. Blending alginate with other polymers (honey, gelatin) [22], amino acids like (polyglutamic acid and poly-l-lysine) [13, 23], and some drugs like N-acetylcysteine (NAC) [24] was studied for improving the erratic degradation, cell-material interaction, cell viability, etc. Jia et al. [6] in their study showed the controlled degradation of oxidized alginate in 3D bioprinting.

Varying biodegradability of solution of sodium alginate along with human adipose stem cells was printed with accurate definition. These kinds of bioinks have the capacity to modulate proliferation and stem cell spreading and withstand uniform cell suspension but are imperfect in the case of stem cell diffusion. Wu et al. [25] showed the procedure of slow degradation of the alginate by tissue incubation in a sodium citrate medium. The sodium citrate amount helped the optimization of the alginate degradation time. Chung et al. [18] improved the printing resolution and printability of pre-crosslinked printed constructs by adding alginate with gelatin, keeping the mechanical property and the growth of cells, and keeping pore diameter constant. In **Figure 3** the procedure of alginate cross-linking with the added cells is shown.

#### **2.2 Wound healing**

Alginate has been used for dressing of the wounds due to its of good conformability, absorptivity, and mild antiseptic properties coupled with biodegradability and nontoxicity and optimal water vapor transmission rate. Alginate-based products like electrospun mat hydrogels and sponges in dressing of wounds are very good substrates for healing of wounds, which include gel-foaming capability as soon as the absorption of the wound exudates and hemostatic capabilities [26]. It is already been mentioned that dressing wounds with alginate improves healing of wounds through monocyte stimulation to harvest higher cytokine levels like tumor necrosis factor-α and interleukin-6 [27]. Near the wound locations, cytokine production creates pro-inflammatory factors that are helpful for wound healing. Because of the existence of endotoxin in the alginate, a huge level of bioactivity

*Alginates - Recent Uses of This Natural Polymer*

alginate-based biomaterials and applications.

**2.1 3D bioprinting**

were capable of degrading, permitted the vascularization, and obtained minimum inflammatory responses after transplantation. Consequently, alginate-based scaffolds can present suitable characteristics as probable cell and drug carriers for tissue regeneration. The next sections explain the clinical and preclinical analysis of

Sodium alginate which is also known as sodium alginate or algin is a naturally extracted less costly polymer from the brown algae cell walls which have intracellular spaces [10]. Alginate is composed of (1-4)-linked β-d-mannuronic (M) and α-l-guluronic acids (G). Alginate is a polyanionic linear block copolymer made of longer M or G blocks, separated by MG regions. Sodium alginate is a kind of polysaccharide which is charged negatively because it is known that materials which are positively charged produce inflammatory response; this allows the biopolymer to support high biocompatibility and cell growth. G blocks enhance the gel structure and M and MG blocks enhance the elasticity, though a high quantity of M blocks could be the reason of immunogenicity [18]. In alginate matrix, with the help of capillary forces, water and other molecules can be trapped. This feature makes alginate hydrogel suitable for bioink designs. The cell density within the bioink will be very high, whereas the shear stress through the extrusion process decreases the cell viability (80–90%). In the case of inkjet bioprinting, the bioinks have lower cell densities (<16 × 106 cells/mL) and are less viscous (<10 mPa s). This technique suggests 90% cell viabilities but, in laser-assisted bioprinting, needs bioinks with viscosities of 1 and 300 mPa s and also requires medium cell densities (108 cells/ mL). In this method, cell viability is very high (>95%). The alginate-based bioink viscosity rests on the alginate molecular weight, alginate concentration, density of cells, and cell phenotype. These are the variables that scientist must consider to optimize the viscosity of the alginate-based bioinks. An additional significant rheological characteristic of aqueous alginate solutions is the shear-thinning, where the shear rate increases while decreasing the viscosity. The viscosity also depends on the performed printing temperature; when the temperature increases, the viscosity gradually decreases. In comparison with other polymers, alginate is convincingly easy to handle and to print and is easy to extrude (printing) while defending the encapsulated cells. Even if it is also a non-cell-adhesive [12], in case of cell encapsulation, alginate is currently one of the most applied biomaterials. After the printing performance, the hydrogel should degrade suitably, allowing the cells to make their specific extra cellular matrix (ECM). The alginate also generates durable insistent cell-laden hydrogels; however oxidation can be performed by slow degradation, for example, sodium peroxide [11]. The main issue of alginate for using it as a biomaterial in bioprinting is slow degradation rate. The release of the hydrogels through the bioprinter nozzle in bioprinting (extrusion) limits the usage to low weight of alginate, which has a major role in the application of reduced mechanical properties. Though the alginate mechanical and structural characteristics are needed for all printed tissue, the biomimicry characteristics required in every instance can be changed by combining new biomaterials in the scaffold or by applying different types of hydrogel fabrication technique. For example, CELLINK is a commercial bioink which is already available for bioprinting; it combines with alginate hydrogel and nanocellulose and presents fast cross-linking and shear-thinning properties; this bioink is appreciated for soft tissue engineering for bioprinting [8]. The formation of blood vessel-like channels is able to transport different materials like nutrients and oxygen via the bioprinted material, which is needed in order to print organs or tissues. To succeed in this aim, Zhang et al. [19] made vessel-like printable

**112**

**Figure 3.** *Alginate with cell cross-linking process.*

is present in these dressings. In situ-forming wound dressing hydrogel can be produced by oxidized alginate and gelatin in low borax concentration as shown by Balakrishnan and Jayakrishnan [28]. The homeostatic gelatin effect is present in the mixed matrix and wound healing property of alginate, and the antiseptic borax property makes alginate the appropriate wound dressing material. Tissueengineered cartilage requirement is immense and has a huge clinical importance. The main causes of disability of the articular cartilage are degenerative and traumatic lesions [29]. Nearly 100 million Chinese people suffer from osteoarthritis. Because of this reason, regeneration and repair of the cartilage have huge impact. The pros of the cartilage repair injectable therapies are that implant within the defect is not only maintained, but it also allows quick bearing of weight because of strength and stiffness which is attained quickly [30, 31]. For bringing close the mechanical properties of the native tissues with the scaffolds, the alginate physical properties are matched with the articular cartilage. Ge and solid alginate injectable hydrogel microspheres are used for cartilage regeneration. Many researchers have studied the growth factor in tissue engineering by using alginate hydrogels and alginate-based microsphere combinations [32, 33]. In one study the demonstration of immobilization of the positive effect of RGD to an alginate porous scaffold for endorsing TGF-β-induced human MSC differentiation is shown [34]. Bian et al. studied the co-encapsulation of the TGF-β including the microsphere of the alginate with the human MSCs in the hyaluronic acid (HA) hydrogels with respect to the design of the constructs implantable for the cartilage repair [35]. The immobilized RGD peptide facilitated the cell-matrix interaction which is proven to be an important feature for the microenvironment of the cells, allowing good cell availability for the chondrogenic-inducing molecule TGF-β. TGF-β-laden alginate microspheres in combination with alginate hydrogels forms a compound carrier which may retain TGF-β bioactivity in the construct and encourages hondrogenesis of MSCs when inserted. The animal experiment displayed that chondrocytes planted into the microsphere scaffold lived habitually in SCID mice and cartilage-like constructions were created after 4 weeks of imbedding.
