**Abstract**

Many soft tissues of the human body such as cartilages, muscles, and ligaments are mainly composed of biological hydrogels possessing excellent mechanical properties and delicate structures. Nowadays, bio-inspired hydrogels have been intensively explored due to their promising potential applications in tissue engineering. However, the traditional manufacturing technology is challenging to produce the bio-inspired hydrogels, and the typical biological composite topologies of bioinspired hydrogels are accessible completed using 3D bioprinting at micrometer resolution. In this chapter, the 3D bioprinting techniques used for the fabrication of bio-inspired hydrogels were summarized, and the materials used were outlined. This chapter also focuses on the applications of bio-inspired hydrogels fabricated using available 3D bioprinting technologies. The development of 3D bioprinting techniques in the future would bring us closer to the fabrication capabilities of living organisms, which would be widely used in biomedical applications.

**Keywords:** 3D bioprinting, hydrogels, biopolymers, tissue engineering, biomaterials

### **1. Introduction**

The design of scaffold materials that can guide tissue regeneration is a very challenging goal [1]. In addition, to support and promote the growth and differentiation of specific cells, an ideal scaffold requires careful control of the material's structure in the range of nanometers to centimeters, and some natural materials with complex structure exist in nature, which provides ideas for the design of ideal scaffolds [2]. These natural materials, such as mammal bones, abalone pearl layers and fish scales, which are composed of multi-layer biominerals and biopolymers, have complex microstructure, which can control the crack growth and fracture in three-dimensional (3D) direction, producing much more strength and toughness than their constituent materials [3–5]. Jellyfish and sea anemones, with a water content of up to 90%, show that their gelatinous bodies exhibit exciting mechanical properties and are able to respond quickly to various environmental stimuli [6–8]. There are also some soft support tissues (such as tendons, ligaments, meniscus, and cartilage), showing softness, toughness and impact resistance [9]. Because of the beneficial properties of natural composite materials, the design of bionic materials has attracted significant attention. Bio-inspired material is considered as a kind of material inspired by nature or biology and then developed by simulating some characteristics [10], and usually, the bio-inspired materials provide better functions than synthetic materials [11].

#### *Biomimetics*

However, there are still many limitations on the fabrication of bio-inspired materials using traditional material manufacturing technology because they cannot accurately control the distribution and spatial trend of micro-holes inside the materials, and it is challenging to produce the contour matching with natural materials [12, 13]. Recently, 3D bioprinting technology has become a promising tool for manufacturing materials with high-precision, which can overcome the limitations compare with the traditional methods, and finally can eventually produce complex and delicate biomimetic 3D structures. Also, 3D bioprinting technology realizes the automatic biological preparation of cell-laden structure through the layered deposition of bio-inks *in vitro* and *in vivo* [14]. In addition, 3D bioprinting technologies are controlled by computers and can be combined with medical imaging systems, such as computed tomography and magnetic resonance imaging (MRI), also combined with computer-aided design (CAD) and computer-aided manufacturing (CAM), to generate personalized structures organized in different length proportions [15, 16]. Compared with the classical tissue engineering methods, 3D bioprinting allows the direct manufacture of complex 3D structures containing spatial variations of biological materials, cells and biochemical substances with the same structure, which significantly improves the biological simulation level of the composition, structure and biochemical characteristics of cell niche in the human body [17]. The complexity of the resulting structure is not only related to the application of tissue regeneration but also to the development of cell biology, drug development, and disease research *in vitro* models [18].

In recent years, in tissue engineering development, many materials have been developed to meet the needs of 3D bioprinting. The most common 3D bioprinting materials are metals, engineering plastics, photosensitive resins, bioplastics and polymer hydrogels. The bio-inspired hydrogels are very similar to natural extracellular matrix (ECM) and display potential advantages in tissue engineering [19]. Bio-inspired hydrogel provides an adequate and porous microenvironment that allows good nutrition and oxygen to diffuse into the encapsulated cells and can be modified to guide cellular processes with various physical, chemical, and biological cues [20]. Besides, these hydrogels are usually non-toxic or low toxic and have good reproducibility. Next, the 3D bioprinting techniques used for the fabrication of bioinspired hydrogels were summarized, and the materials used for 3D bioprinting were outlined. This chapter also focuses on the applications of bio-inspired hydrogels.

### **2. 3D bioprinting techniques**

There are several available 3D bioprinting techniques for fabricating bioinspired hydrogels, including inkjet bioprinting, laser-assisted bioprinting, extrusion bioprinting, and stereolithography, as shown in **Figure 1** [21].

#### **2.1 Inkjet bioprinting**

During the inkjet bioprinting process, biomaterials are selectively placed on the construction platform layer by layer until the required structure is formed. The first inkjet printers for bioprinting applications were improved versions of commercial two-dimensional ink printers [22]. For the inkjet bioprinting, the ink in the ink cartridge is replaced by biomaterials, and the paper is replaced by an electronically controlled lifting table to provide the control of the third dimension Z-axis in addition to the X-and Y-axes. The bioprinter based on inkjet printing technology is customized to process and print biomaterials with higher resolution, accuracy and speed [16]. Inkjet bioprinters use thermal or acoustic forces to spray droplets

**3**

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

network in the whole hydrogel [26].

**2.2 Laser-assisted bioprinting**

engineering [27].

**Figure 1.**

onto the substrate, which can support or form part of the final structure [23]. Thermal inkjet uses a heating element to induce the evaporation of a small volume of bioink in a reservoir, thereby forming and ejecting a small droplet. Therefore, in the printing process, this method keeps the cells at high temperature (300°C) for several microseconds (about 2 microseconds), which may lead to the formation of transient pores in the cell membrane [16]. Using the thermal inkjet printer, Solis et al., studied the effect of heat generated by the thermal ink-jet bio printer and found that the survival rate of Chinese hamster ovary (CHO) cells was 89% [24]. Such survival rate of cells could be greatly improved by using a piezoelectric inkjet printer, the generation and injection of droplets are realized by applying external voltage to control the mechanical deformation of piezoelectric transducer, which prevents the temperature from rising to the super physiological level [25]. Compaan et al. used alginate as the sacrificial material to prepare cell-supported silk fibroin hydrogels with a clear structure based on the piezoelectric inkjet 3D bioprinting system. The printed tubular structure has a diameter of 5 mm, a height of 2.5 or 5.0 mm and a thickness of about 400 microns. Moreover, the effect of citrate treatment on the printing was compared. The results showed that alginate removal and alginate removal could enable cells to extend and contact each other and form a cell

*Bioprinting techniques mainly include inkjet, laser-assisted, extrusion and stereolithography [21].*

The advantages of inkjet bioprinting mainly include: low cost due to its similar structure to commercial printers, high printing speed due to the ability of the print head to support parallel operation mode, and relatively high unit survival rate (usually from 80–90%) determined by many experimental results. However, the risks of cells and materials exposed to thermal and mechanical stresses, low droplet directionality, uneven droplet size, frequent nozzle plugging, and unreliable cell encapsulation have brought considerable limitations to the application in tissue

The typical laser-assisted biological printing device include pulsed laser beams, focusing systems, and donor bands that respond to laser stimuli, consisting of glass covered with laser energy absorbing layers, and biomaterial layers (such as cells/ hydrogel composite) prepared in liquid and receiving substrates for ribbons. The principle of laser-assisted bioprinting is to apply high-energy pulse laser (usually near-infrared laser) to the donor color band coated with bioink. This laser pulse evaporates a part of the donor layer, forms a high-pressure bubble on the interface of the bioink layer, and pushes the materials containing cells to the receiving

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

**Figure 1.**

*Biomimetics*

disease research *in vitro* models [18].

**2. 3D bioprinting techniques**

**2.1 Inkjet bioprinting**

However, there are still many limitations on the fabrication of bio-inspired materials using traditional material manufacturing technology because they cannot accurately control the distribution and spatial trend of micro-holes inside the materials, and it is challenging to produce the contour matching with natural materials [12, 13]. Recently, 3D bioprinting technology has become a promising tool for manufacturing materials with high-precision, which can overcome the limitations compare with the traditional methods, and finally can eventually produce complex and delicate biomimetic 3D structures. Also, 3D bioprinting technology realizes the automatic biological preparation of cell-laden structure through the layered deposition of bio-inks *in vitro* and *in vivo* [14]. In addition, 3D bioprinting technologies are controlled by computers and can be combined with medical imaging systems, such as computed tomography and magnetic resonance imaging (MRI), also combined with computer-aided design (CAD) and computer-aided manufacturing (CAM), to generate personalized structures organized in different length proportions [15, 16]. Compared with the classical tissue engineering methods, 3D bioprinting allows the direct manufacture of complex 3D structures containing spatial variations of biological materials, cells and biochemical substances with the same structure, which significantly improves the biological simulation level of the composition, structure and biochemical characteristics of cell niche in the human body [17]. The complexity of the resulting structure is not only related to the application of tissue regeneration but also to the development of cell biology, drug development, and

In recent years, in tissue engineering development, many materials have been developed to meet the needs of 3D bioprinting. The most common 3D bioprinting materials are metals, engineering plastics, photosensitive resins, bioplastics and polymer hydrogels. The bio-inspired hydrogels are very similar to natural extracellular matrix (ECM) and display potential advantages in tissue engineering [19]. Bio-inspired hydrogel provides an adequate and porous microenvironment that allows good nutrition and oxygen to diffuse into the encapsulated cells and can be modified to guide cellular processes with various physical, chemical, and biological cues [20]. Besides, these hydrogels are usually non-toxic or low toxic and have good reproducibility. Next, the 3D bioprinting techniques used for the fabrication of bioinspired hydrogels were summarized, and the materials used for 3D bioprinting were outlined. This chapter also focuses on the applications of bio-inspired hydrogels.

There are several available 3D bioprinting techniques for fabricating bioinspired hydrogels, including inkjet bioprinting, laser-assisted bioprinting, extru-

During the inkjet bioprinting process, biomaterials are selectively placed on the construction platform layer by layer until the required structure is formed. The first inkjet printers for bioprinting applications were improved versions of commercial two-dimensional ink printers [22]. For the inkjet bioprinting, the ink in the ink cartridge is replaced by biomaterials, and the paper is replaced by an electronically controlled lifting table to provide the control of the third dimension Z-axis in addition to the X-and Y-axes. The bioprinter based on inkjet printing technology is customized to process and print biomaterials with higher resolution, accuracy and speed [16]. Inkjet bioprinters use thermal or acoustic forces to spray droplets

sion bioprinting, and stereolithography, as shown in **Figure 1** [21].

**2**

*Bioprinting techniques mainly include inkjet, laser-assisted, extrusion and stereolithography [21].*

onto the substrate, which can support or form part of the final structure [23]. Thermal inkjet uses a heating element to induce the evaporation of a small volume of bioink in a reservoir, thereby forming and ejecting a small droplet. Therefore, in the printing process, this method keeps the cells at high temperature (300°C) for several microseconds (about 2 microseconds), which may lead to the formation of transient pores in the cell membrane [16]. Using the thermal inkjet printer, Solis et al., studied the effect of heat generated by the thermal ink-jet bio printer and found that the survival rate of Chinese hamster ovary (CHO) cells was 89% [24]. Such survival rate of cells could be greatly improved by using a piezoelectric inkjet printer, the generation and injection of droplets are realized by applying external voltage to control the mechanical deformation of piezoelectric transducer, which prevents the temperature from rising to the super physiological level [25]. Compaan et al. used alginate as the sacrificial material to prepare cell-supported silk fibroin hydrogels with a clear structure based on the piezoelectric inkjet 3D bioprinting system. The printed tubular structure has a diameter of 5 mm, a height of 2.5 or 5.0 mm and a thickness of about 400 microns. Moreover, the effect of citrate treatment on the printing was compared. The results showed that alginate removal and alginate removal could enable cells to extend and contact each other and form a cell network in the whole hydrogel [26].

The advantages of inkjet bioprinting mainly include: low cost due to its similar structure to commercial printers, high printing speed due to the ability of the print head to support parallel operation mode, and relatively high unit survival rate (usually from 80–90%) determined by many experimental results. However, the risks of cells and materials exposed to thermal and mechanical stresses, low droplet directionality, uneven droplet size, frequent nozzle plugging, and unreliable cell encapsulation have brought considerable limitations to the application in tissue engineering [27].

#### **2.2 Laser-assisted bioprinting**

The typical laser-assisted biological printing device include pulsed laser beams, focusing systems, and donor bands that respond to laser stimuli, consisting of glass covered with laser energy absorbing layers, and biomaterial layers (such as cells/ hydrogel composite) prepared in liquid and receiving substrates for ribbons. The principle of laser-assisted bioprinting is to apply high-energy pulse laser (usually near-infrared laser) to the donor color band coated with bioink. This laser pulse evaporates a part of the donor layer, forms a high-pressure bubble on the interface of the bioink layer, and pushes the materials containing cells to the receiving

substrate [16, 25, 28]. Compared with inkjet bioprinting, laser-assisted bioprinting can avoid the problem of jamming cell or material, also can avoid direct contact with the printer and biological ink at the same time. The non-contact biological printing method can choose much more types of ink, resulting in printing materials with wider range of viscosity [28].

The laser pulse energy, ECM thickness, and bioink viscosity can influence cell viability. The higher the laser energy is, the higher the cell death rate is, but the increase of membrane thickness and bioink viscosity will lead to an increase of cell viability. Guillotin et al. studied the effects of bioink viscosity, laser energy and printing speed on printing resolution. The microscale resolution and 5 kHz printing speed could be achieved, and the laser-assisted bioprinting could combine cells with ECM to produce soft tissue with high cell density *in vivo* [29]. Laser-assisted biological printing is considered as one of the most promising methods to fabricate engineered tissue because of its unique resolution, high throughput, high resolution, and high resolution, as well as the ability to produce heterogeneous tissue structures with high cell density [25]. However, compared with other bioprinting methods, the laser diode with high resolution and high intensity are expensive, and the control of the laser printing system is complex, which limit the application of this technology [28].

#### **2.3 Extrusion bioprinting**

The extrusion bioprinting can fabricate 3D cell carriers for tissue regeneration. The prepolymer solutions need to be prepared first, and almost all types of prepolymer solutions with different viscosities and aggregates with high cell density can be printed with extruded bioprinters [28]. Different from printing small droplets onto the platform, the extrusion bioprinting continuously deposit hydrogel filaments within a diameter of 150–300 microns to generate 3D structures. Common extrusion bioprinting method includes pneumatic, piston-driven, and screw-driven dispensing. In pneumatic dispensing, air pressure provides the required driving force, while in piston and screw-driven dispensing, vertical and rotating mechanical forces start printing respectively [30]. There are three main factors that decide the printability of extrusion bioprinting, mainly including the adjustability of viscosity, the bioink phase before extrusion, and the material-specific bio-manufacturing window [31]. Extrusion bioprinters have been used to produce various tissue types, such as aortic valves, branching vascular trees, in vitro drug movement and tumor models [32]. Although the manufacturing time may be prolonged for high-resolution complex structures, the structures have been manufactured from the clinically related tissue size to the microtissue in the microfluidic chamber. Furthermore, it is convenient to combine cells with bioactive agents, because that the heating process is not involved [33]. Compared with inkjet 3D bioprinting, extrusion bioprinters can achieve a continuous flow of biomaterials, thus achieving the simplicity of operation and a broader selection of biomaterials, including polymers, acellular matrices, cellular hydrogels, spheres and aggregates [34].

#### **2.4 Stereolithography**

Among all the bioprinting technologies, stereolithography (SLA) 3D bioprinting display much more advantages over extrusion or ink-jet bioprinting technology [28]. SLA is based on the polymerization of photosensitive polymers, and the digital mirror array controls the light band in the projection field to achieve selective crosslinking of each layer of the hydrogel prepolymer solution [35]. No matter how intricate a layer's pattern is, the printing time is the same because the whole pattern is projected on the printing plane. Therefore, the printer only needs a movable table

**5**

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

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

**3.1 Natural polymers**

in the vertical direction, which significantly simplifies the control of the printer. The cell encapsulated scaffold fabricated by the SLA system can achieve 100 μm resolution with printing time less than 1 hour, also maintain very high cell viability (90%) [36]. The above properties make SLA practical for fabricating delicate construct for tissue engineering. Arcaute et al. used composite lithography technology and two different molecular weight of polyethylene glycol (PEG) to prepare composite multilayer 3D structure of PEG hydrogel, and the properties of prepared hydrogel were influenced by photo-initiator and photosensitive polymer concentration. Besides, the prepared PEG hydrogel supports attachment, proliferation and differentiation of bovine chondrocytes, providing evidence for the applicability of resins for cartilage tissue engineering [37]. Valentin et al. prepared the sodium alginate precursor solution based on ion crosslinking, and different concentrations of cationic sources, such as barium carbonate, magnesium carbonate and calcium carbonate, and photo acid generator (PAG), diphenyliodonium nitrate were used, and the sodium alginate hydrogel was printed by SLA. The printed alginate hydrogel exhibited different mechanical and physical properties when crosslinked with two kinds of cations. The microstructures with variable height could be printed with optimized precursor formulations. Due to the high resolution, the 3D fabrication of natural and synthetic polyelectrolyte hydrogels via SLA enables lab-on-a-chip devices, soft sensors and actuators, and other biologically-inspired devices [38].

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].

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 interac-

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 proper-

ties, 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

tion with cells to promote cell proliferation and differentiation.

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

*Biomimetics*

with wider range of viscosity [28].

**2.3 Extrusion bioprinting**

**2.4 Stereolithography**

substrate [16, 25, 28]. Compared with inkjet bioprinting, laser-assisted bioprinting can avoid the problem of jamming cell or material, also can avoid direct contact with the printer and biological ink at the same time. The non-contact biological printing method can choose much more types of ink, resulting in printing materials

The laser pulse energy, ECM thickness, and bioink viscosity can influence cell viability. The higher the laser energy is, the higher the cell death rate is, but the increase of membrane thickness and bioink viscosity will lead to an increase of cell viability. Guillotin et al. studied the effects of bioink viscosity, laser energy and printing speed on printing resolution. The microscale resolution and 5 kHz printing speed could be achieved, and the laser-assisted bioprinting could combine cells with ECM to produce soft tissue with high cell density *in vivo* [29]. Laser-assisted biological printing is considered as one of the most promising methods to fabricate engineered tissue because of its unique resolution, high throughput, high resolution, and high resolution, as well as the ability to produce heterogeneous tissue structures with high cell density [25]. However, compared with other bioprinting methods, the laser diode with high resolution and high intensity are expensive, and the control of the laser printing system is complex, which limit the application of this technology [28].

The extrusion bioprinting can fabricate 3D cell carriers for tissue regeneration. The prepolymer solutions need to be prepared first, and almost all types of prepolymer solutions with different viscosities and aggregates with high cell density can be printed with extruded bioprinters [28]. Different from printing small droplets onto the platform, the extrusion bioprinting continuously deposit hydrogel filaments within a diameter of 150–300 microns to generate 3D structures. Common extrusion bioprinting method includes pneumatic, piston-driven, and screw-driven dispensing. In pneumatic dispensing, air pressure provides the required driving force, while in piston and screw-driven dispensing, vertical and rotating mechanical forces start printing respectively [30]. There are three main factors that decide the printability of extrusion bioprinting, mainly including the adjustability of viscosity, the bioink phase before extrusion, and the material-specific bio-manufacturing window [31]. Extrusion bioprinters have been used to produce various tissue types, such as aortic valves, branching vascular trees, in vitro drug movement and tumor models [32]. Although the manufacturing time may be prolonged for high-resolution complex structures, the structures have been manufactured from the clinically related tissue size to the microtissue in the microfluidic chamber. Furthermore, it is convenient to combine cells with bioactive agents, because that the heating process is not involved [33]. Compared with inkjet 3D bioprinting, extrusion bioprinters can achieve a continuous flow of biomaterials, thus achieving the simplicity of operation and a broader selection of biomaterials, including polymers, acellular

Among all the bioprinting technologies, stereolithography (SLA) 3D bioprinting

display much more advantages over extrusion or ink-jet bioprinting technology [28]. SLA is based on the polymerization of photosensitive polymers, and the digital mirror array controls the light band in the projection field to achieve selective crosslinking of each layer of the hydrogel prepolymer solution [35]. No matter how intricate a layer's pattern is, the printing time is the same because the whole pattern is projected on the printing plane. Therefore, the printer only needs a movable table

matrices, cellular hydrogels, spheres and aggregates [34].

**4**

in the vertical direction, which significantly simplifies the control of the printer. The cell encapsulated scaffold fabricated by the SLA system can achieve 100 μm resolution with printing time less than 1 hour, also maintain very high cell viability (90%) [36]. The above properties make SLA practical for fabricating delicate construct for tissue engineering. Arcaute et al. used composite lithography technology and two different molecular weight of polyethylene glycol (PEG) to prepare composite multilayer 3D structure of PEG hydrogel, and the properties of prepared hydrogel were influenced by photo-initiator and photosensitive polymer concentration. Besides, the prepared PEG hydrogel supports attachment, proliferation and differentiation of bovine chondrocytes, providing evidence for the applicability of resins for cartilage tissue engineering [37]. Valentin et al. prepared the sodium alginate precursor solution based on ion crosslinking, and different concentrations of cationic sources, such as barium carbonate, magnesium carbonate and calcium carbonate, and photo acid generator (PAG), diphenyliodonium nitrate were used, and the sodium alginate hydrogel was printed by SLA. The printed alginate hydrogel exhibited different mechanical and physical properties when crosslinked with two kinds of cations. The microstructures with variable height could be printed with optimized precursor formulations. Due to the high resolution, the 3D fabrication of natural and synthetic polyelectrolyte hydrogels via SLA enables lab-on-a-chip devices, soft sensors and actuators, and other biologically-inspired devices [38].
