**3. Polymers used for bio-inspired hydrogels**

Hydrogels are considered as the gold standard materials for 3D bioprinting because they can provide a flexible and hydrated cross-linked network, similar to the natural extracellular matrix, in which cells can survive [39]. The polymers prepared for hydrogels can be classified into natural and synthetic polymers [40]. The natural polymers include alginate, chitosan, hyaluronic acid, gelatin, and so on, and the synthetic polymers mainly include polyacrylamide (PAAm), polyvinyl alcohol (PVA), polyethylene glycol (PEG), polylactic acid (PLA), and so on [41, 42].

#### **3.1 Natural polymers**

Most hydrogels prepared by natural polymers have the advantages of good hydrophilicity, good biocompatibility, specific enzymatic degradation, and contain various active functional groups and structural domains, and display better interaction with cells to promote cell proliferation and differentiation.

Alginate is extracted from alginate plants, is a kind of natural high molecular, composing of β-d-mannuronate (M) and α-l-guluronate (G). Alginate has been widely used in tissue engineering because of its advantages of abundant production, low price, good biocompatibility, and abundant functional groups, which are suitable for the preparation of bioink for 3D bioprinting [43, 44]. Alginate can react with CaCO3 to release bivalent Ca2+ and then form an ionic crosslinking hydrogel bonded with -COO- on G unit of alginate G unit, to achieve the controllability of alginate ion crosslinking. The alginate hydrogel has high toughness and good mechanical properties, but the degradation rate of the alginate hydrogel is not controllable [45].

Chitosan is the product of deacetylation of chitin, which has a straight-chain structure and positive charge due to the presence of amino groups. Because of the useful biological function and biocompatibility, the degradation by microorganisms, chitosan has been widely concerned and applied in various industries [46]. The chitosan ink can be directly printed in air, and then the chitosan scaffold is refined by physical gelation. A chitosan hydrogel that satisfies both biocompatibility and mechanical properties has been obtained, and it has been confirmed that chitosan hydrogel can guide cell growth [47].

Gelatin is the hydrolysate of collagen, which contains many arginine-glycineaspartic-acid (RGD) sequences and matrix metalloproteinase (MMP) target sequences, which enhance cell adhesion and cellular microenvironment remodeling respectively [48]. Because of biodegradability, biocompatibility, and low antigenicity, gelatin is attractive for bio-inspired hydrogel [49]. Lewis et al. used gelatin as a bioink to print into a specific 3D geometry using 3D bioprinting, which can regulate the biological processes of hepatocytes, enhance protein function, and facilitate cell proliferation and differentiation [50]. Another commonly used gelatin derivative is to acylate gelatin to form gelatin methacrylamide (GelMA) [51]. Zhou et al. used GelMA, N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy) butanamide linked hyaluronic acid (HA-NB) and photo-initiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as biomimetic bioink to fabricated a bio-inspired 3D tissue construct via the digital light process (DLP)-based 3D bioprinting technology for ski regeneration (**Figure 2**) [52]. Bhise et al. used GelMA to carry out Hep G2/C3A cells to prepare biomimetic 3D liver structure hydrogel through bioprinting technology. A bionic human body chip of liver tissue was prepared by bioreactor. The toxicity response test of this chip in the test of acetaminophen is similar to that reported *in vivo* and other *in vitro* models, so this provides conceptual proof that the liver biomimetic human chip can be used in vitro drug toxicity screening experiments [53].

Hyaluronic acid (HA) is a kind of biocompatible non-sulfated glycosaminoglycan composed of N-acetylglucosamine and D-glucuronic acid repeated disaccharide units [54]. It is abundant in tissues including cartilage, neurons and skin. HA is of intrinsic biological importance because it binds to receptors such as CD44, can be degraded by oxidative species and hyaluronidase, and is related to the function and structure of development, wound healing and adult tissues. Because of biocompatibility, biodegradability, and natural biological function, HA hydrogels are widely used in various application fields [55]. Besides, the HA hydrogel can energize cell viability and promote osteoblasts to differentiate into cartilage. Unlike collagen and other proteins, the sequence of HA is different from species and its antigenicity is low, so it is especially promising as an injectable hydrogel.

Several other natural polymers, such as collagen, agarose, carrageenan, fibrin, heparin, chondroitin sulfate, cellulose, hemicellulose, lignin, and so on, could be used for hydrogels using 3D bioprinting [21]. However, natural hydrogels lack adequate mechanical properties, especially when implanted *in vivo* for a long time. Because of the uncontrollable swelling in physiological water environment, the mechanical stability of scaffolds tends to decrease. Thus, the chemical modification on natural polymers would be necessary to improve their printability as bioink, and the pending chemical groups after medication will improve the mechanical properties of construct after 3D bioprinting.

#### **3.2 Synthetic polymers**

The hydrogels fabricated using synthetic polymers have the advantages of long service life, strong water absorption, and high gel strength [41]. Polyacrylamide (PA) is a general designation of acrylamide homopolymer and copolymer. PA is a kind of water-soluble polymer, which has many amide groups in its structure and is easy to form hydrogen bond, so it has good stability and flocculation and is easy to be chemically modified. Ahn et al. grafted poly (N-isopropylacrylamide) (PNIPAAm) onto the framework of sodium alginate and synthesized sodium alginate PNIPAAm polymer micelles by self-assembly in aqueous solution, and the micelles could be used for the encapsulation of anticancer drug adriamycin [56]. Polyethylene glycol

**7**

**Figure 2.**

*Compressive Young's modulus [52].*

*Bio-Inspired Hydrogels via 3D Bioprinting DOI: http://dx.doi.org/10.5772/intechopen.94985*

(PEG) is another synthetic polymer, and it has no toxicity and irritation, has good biocompatibility, and can be discharged from the body through the kidney. It has been widely used in the field of biomedicine [57]. Gao et al. constructed the polyethylene glycol diacrylate (PEGDA) hydrogel with uniform distribution of human mesenchymal stem cells (hMSCs) inside by simultaneous photopolymerization with commercial thermojet printers. hMSCs filled in 3D PEGDA hydrogel showed no deposition during culture and showed a chondrogenic phenotype [58]. Wang et al. prepared an injectable hydrogel through *in situ* Michael addition reaction between tetraniline-polyethylene glycol diacrylate (TA-PEG) and thiol hyaluronic acid (HA-

*(A) Fabrication of rapid gelation and tough GelMA/HA-NB/LAP hydrogel for DLP-based printing. (B) the skin analogous with sophisticated two-layer gel structure was fabricated via 3D bioprinting. (a) the bioink was printed with a layer-by-layer style using a DLP-based 3D printer. (b, c) the structure of native skin was displayed in CAD images. (d) the lower layer view of the scaffold was shown. (e) CAD images of different designed microchannel size and the printed products. (f) the elastic compressibility of products. (g)* 

Polylactic acid (PLA) is a kind of polymer, which is made of lactic acid as the primary raw material, and through polymerization, in which the performance can be adjusted by the structure [60]. Senatov et al. prepared PLA/hyaluronic acid

SH), which was used to carry adipose-derived stem cells (ADSCs) [59].

*Bio-Inspired Hydrogels via 3D Bioprinting DOI: http://dx.doi.org/10.5772/intechopen.94985*

*Biomimetics*

and mechanical properties has been obtained, and it has been confirmed that

Gelatin is the hydrolysate of collagen, which contains many arginine-glycineaspartic-acid (RGD) sequences and matrix metalloproteinase (MMP) target sequences, which enhance cell adhesion and cellular microenvironment remodeling respectively [48]. Because of biodegradability, biocompatibility, and low antigenicity, gelatin is attractive for bio-inspired hydrogel [49]. Lewis et al. used gelatin as a bioink to print into a specific 3D geometry using 3D bioprinting, which can regulate the biological processes of hepatocytes, enhance protein function, and facilitate cell proliferation and differentiation [50]. Another commonly used gelatin derivative is to acylate gelatin to form gelatin methacrylamide (GelMA) [51]. Zhou et al. used GelMA, N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy) butanamide linked hyaluronic acid (HA-NB) and photo-initiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as biomimetic bioink to fabricated a bio-inspired 3D tissue construct via the digital light process (DLP)-based 3D bioprinting technology for ski regeneration (**Figure 2**) [52]. Bhise et al. used GelMA to carry out Hep G2/C3A cells to prepare biomimetic 3D liver structure hydrogel through bioprinting technology. A bionic human body chip of liver tissue was prepared by bioreactor. The toxicity response test of this chip in the test of acetaminophen is similar to that reported *in vivo* and other *in vitro* models, so this provides conceptual proof that the liver biomimetic human chip can be used in vitro drug toxicity screening experiments [53]. Hyaluronic acid (HA) is a kind of biocompatible non-sulfated glycosaminoglycan composed of N-acetylglucosamine and D-glucuronic acid repeated disaccharide units [54]. It is abundant in tissues including cartilage, neurons and skin. HA is of intrinsic biological importance because it binds to receptors such as CD44, can be degraded by oxidative species and hyaluronidase, and is related to the function and structure of development, wound healing and adult tissues. Because of biocompatibility, biodegradability, and natural biological function, HA hydrogels are widely used in various application fields [55]. Besides, the HA hydrogel can energize cell viability and promote osteoblasts to differentiate into cartilage. Unlike collagen and other proteins, the sequence of HA is different from species and its antigenicity is

chitosan hydrogel can guide cell growth [47].

low, so it is especially promising as an injectable hydrogel.

ties of construct after 3D bioprinting.

**3.2 Synthetic polymers**

Several other natural polymers, such as collagen, agarose, carrageenan, fibrin, heparin, chondroitin sulfate, cellulose, hemicellulose, lignin, and so on, could be used for hydrogels using 3D bioprinting [21]. However, natural hydrogels lack adequate mechanical properties, especially when implanted *in vivo* for a long time. Because of the uncontrollable swelling in physiological water environment, the mechanical stability of scaffolds tends to decrease. Thus, the chemical modification on natural polymers would be necessary to improve their printability as bioink, and the pending chemical groups after medication will improve the mechanical proper-

The hydrogels fabricated using synthetic polymers have the advantages of long service life, strong water absorption, and high gel strength [41]. Polyacrylamide (PA) is a general designation of acrylamide homopolymer and copolymer. PA is a kind of water-soluble polymer, which has many amide groups in its structure and is easy to form hydrogen bond, so it has good stability and flocculation and is easy to be chemically modified. Ahn et al. grafted poly (N-isopropylacrylamide) (PNIPAAm) onto the framework of sodium alginate and synthesized sodium alginate PNIPAAm polymer micelles by self-assembly in aqueous solution, and the micelles could be used for the encapsulation of anticancer drug adriamycin [56]. Polyethylene glycol

**6**

#### **Figure 2.**

*(A) Fabrication of rapid gelation and tough GelMA/HA-NB/LAP hydrogel for DLP-based printing. (B) the skin analogous with sophisticated two-layer gel structure was fabricated via 3D bioprinting. (a) the bioink was printed with a layer-by-layer style using a DLP-based 3D printer. (b, c) the structure of native skin was displayed in CAD images. (d) the lower layer view of the scaffold was shown. (e) CAD images of different designed microchannel size and the printed products. (f) the elastic compressibility of products. (g) Compressive Young's modulus [52].*

(PEG) is another synthetic polymer, and it has no toxicity and irritation, has good biocompatibility, and can be discharged from the body through the kidney. It has been widely used in the field of biomedicine [57]. Gao et al. constructed the polyethylene glycol diacrylate (PEGDA) hydrogel with uniform distribution of human mesenchymal stem cells (hMSCs) inside by simultaneous photopolymerization with commercial thermojet printers. hMSCs filled in 3D PEGDA hydrogel showed no deposition during culture and showed a chondrogenic phenotype [58]. Wang et al. prepared an injectable hydrogel through *in situ* Michael addition reaction between tetraniline-polyethylene glycol diacrylate (TA-PEG) and thiol hyaluronic acid (HA-SH), which was used to carry adipose-derived stem cells (ADSCs) [59].

Polylactic acid (PLA) is a kind of polymer, which is made of lactic acid as the primary raw material, and through polymerization, in which the performance can be adjusted by the structure [60]. Senatov et al. prepared PLA/hyaluronic acid

(HA) interconnected porous scaffold via a melt-wire method; the 3D printing technique avoided thermal degradation of PLA, the porosity and pore size of the scaffold could be well controlled. The porous PLA/HA scaffold with 15% HA has a considerable crack resistance and can work for a long time under the stress of 21 MPa, which was potential for bone tissue engineering applications [61].

Polyvinyl alcohol (PVA) is a synthetic water-soluble polymer, it has good biodegradation, biocompatibility, and no side effects on the human body [62]. PVA has been widely used in ophthalmology, wound dressing, artificial joint, and so on [42, 63]. Shi et al. prepared an injectable dynamic hydrogel using HA grafted with PVA and phenyl boric acid (PBA). The synthesized HA-PBA-PVA dynamic hydrogel has the reactive oxygen species reactivity and the scavenging activity of active oxygen. Furthermore, the hydrogel had good biocompatibility to the encapsulated neural precursor cells (NPC), and its ability to scavenge reactive oxygen species could protect the NPC cells from the damage of reactive oxygen species. The HA-PBA-PVA hydrogel could be used as bioink for 3D biological printing to prepare multilayer and cell loaded structures. The NPC cells showed good viability (85 ± 2% of living cells) after extrusion and maintained the excellent viability of 81 ± 2% of living cells after 3 days of culture. The results indicated that multifunctional injectable and ROS responsive self-healing HA-PBA-PVA dynamic hydrogels were expected to be candidates for 3D culture and 3D bioprinting [64].

Besides, there are also many other synthetic polymers for the fabrication of bio-inspired hydrogels, such as Pluronic and derivatives, PEG or polyethylene oxide (PEO) based block copolymers, poly(L-glutamic acid), poly(propylene fumarate), methoxy polyethylene glycol, and so on. Though, the synthetic polymers can precisely control their gel structure and properties and have better physical and chemical stability and more raw materials to prepare bio-inspired hydrogels. However, it is necessary to pay attention to the possible biocompatibility of unreacted monomers and residual initiators during the preparation of synthetic polymer materials, and the biocompatibility could be greatly improved via compositing or liking with natural polymers [65–67].
