**2. 3D bioprinting for designing bioscaffold**

The conventional method to develop an engineered-tissue product involved the initial fabrication of specific native tissue design followed by the provision of cells and biomolecules. However, this approach could contribute to two major drawbacks including limitation in cell distribution and reduction in cell growth due to low nutrient concentration at the core area [10]. Very commonly used techniques for fabricating 3D scaffolds include freeze casting, solvent casting, gas foaming, and salt leaching [18]. The technology advancement in tissue engineering has been contributed to the current approach through computer-aided layered manufacturing technique, which is also known as 3D bioprinting. Briefly, the 3D bioprinting technology involves the combination of the primary ingredients known as "bio-ink" that functions as a biological framework and various types of cells with the presence of chemical factors, and biomolecules to form a solid and functional *in situ* 3D living structure [19].

There are four different techniques under 3D bioprinting including inkjet printing, extrusion-based methods, light-induced (photopolymerization) methods and particle fusion-based methods [7, 20–27]. The first three abovementioned techniques have been widely used to fabricate biomaterial designs [7].

The inkjet-based 3D bioprinting (**Figure 1**), first developed by Thomas Boland from Clemson University in 2003, is a low-cost manufacturing process that performs high-speed printing for 3D structure [21]. Besides, it provides high-resolution printing output up to 50 μm and widely proven to support cell viability and growth [22]. However, the main drawbacks are dealing with a low concentration of printing ink could hamper the reliability of cell encapsulation and significantly affect print fidelity [23]. Besides, this approach potentially could damage the printed

**189**

*3D Printed Bioscaffolds for Developing Tissue-Engineered Constructs*

cells on the plantar under shear stress created through the inkjet-based printing but no concrete evidence is reported so far [21, 24]. Three main stages could affect the printable ink such as the production of the droplet, droplet/substrate closeinteraction, and polymerization of the droplet. The two mechanisms, which have been involved under droplet generation through inkjet-based 3D bioprinting, are drop-on-demand and continuous inkjet [25]. The size of ink droplets produced via drop-on-demand and continuous injection is in the range of 25–50 and 100 μm, respectively [19]. Drop-on-demand inkjet has been conveniently used for tissue

*The inkjet-based 3D bioprinting provides high resolution of printing output around 50 μm.*

The inkjet-based technology can be categorized into three as follows: thermalbased, piezoelectric-based, and magnetic-based inkjet printing [26]. The thermal induction can reach until 100–300°C that is required to nucleate a bubble and directly increase the appropriate pressure in the printhead lead to droplet expulsion [28]. There is no dead effect on the cells due to the presence of high temperature only for a microsecond and the previous study demonstrated consistency in cell viability post-inkjet-based 3D bioprinting [29]. Besides, the ink drop production can be induced by a piezoelectric method that focuses on the pulse pressure or acoustic waves generated from a piezoelectric actuator to expel printing ink drop. Another method to generate the drop expulsion is by using the electromagnetic approach depending on the Lorentz force and permanent magnet-based configurations. However, it produces a larger size of ink droplets as compared to thermal-

The second approach of 3D bioprinting is the extrusion-based method (**Figure 2**) that can be divided into two types consisting of fused deposition modeling and direct ink writing [19]. It is easy to handle, customized-based design bioprinter, and versatile with the developed current system. The principle of this 3D bioprinting method is that the printed ink extruded from the nozzle in liquid or molten state forms a particular line on the platform before polymerizing [30]. The bioprinting ink is commonly in the form of solid coil or filament that goes through the hot nozzle (temperature of around 200°C) before extrusion onto the platform. The extrusion from the printing nozzle is controlled by a specific system using various interventions including pressure-based control, pneumatic or mechanical control, or solenoid control before forming layered printed ink as required by the computerized set up to build up the 3D biomaterial designs [7]. The biopolymer should have an excellent solid-to-melt transition property to produce high-resolution 3D cell-laden on the printer platform [31]. However, the extrusion-based 3D bioprinting potentially could generate high mechanical force

*DOI: http://dx.doi.org/10.5772/intechopen.92418*

engineering applications.

**Figure 1.**

based and piezoelectric-based approaches [28].

*3D Printed Bioscaffolds for Developing Tissue-Engineered Constructs DOI: http://dx.doi.org/10.5772/intechopen.92418*

#### **Figure 1.**

*The inkjet-based 3D bioprinting provides high resolution of printing output around 50 μm.*

cells on the plantar under shear stress created through the inkjet-based printing but no concrete evidence is reported so far [21, 24]. Three main stages could affect the printable ink such as the production of the droplet, droplet/substrate closeinteraction, and polymerization of the droplet. The two mechanisms, which have been involved under droplet generation through inkjet-based 3D bioprinting, are drop-on-demand and continuous inkjet [25]. The size of ink droplets produced via drop-on-demand and continuous injection is in the range of 25–50 and 100 μm, respectively [19]. Drop-on-demand inkjet has been conveniently used for tissue engineering applications.

The inkjet-based technology can be categorized into three as follows: thermalbased, piezoelectric-based, and magnetic-based inkjet printing [26]. The thermal induction can reach until 100–300°C that is required to nucleate a bubble and directly increase the appropriate pressure in the printhead lead to droplet expulsion [28]. There is no dead effect on the cells due to the presence of high temperature only for a microsecond and the previous study demonstrated consistency in cell viability post-inkjet-based 3D bioprinting [29]. Besides, the ink drop production can be induced by a piezoelectric method that focuses on the pulse pressure or acoustic waves generated from a piezoelectric actuator to expel printing ink drop. Another method to generate the drop expulsion is by using the electromagnetic approach depending on the Lorentz force and permanent magnet-based configurations. However, it produces a larger size of ink droplets as compared to thermalbased and piezoelectric-based approaches [28].

The second approach of 3D bioprinting is the extrusion-based method (**Figure 2**) that can be divided into two types consisting of fused deposition modeling and direct ink writing [19]. It is easy to handle, customized-based design bioprinter, and versatile with the developed current system. The principle of this 3D bioprinting method is that the printed ink extruded from the nozzle in liquid or molten state forms a particular line on the platform before polymerizing [30]. The bioprinting ink is commonly in the form of solid coil or filament that goes through the hot nozzle (temperature of around 200°C) before extrusion onto the platform. The extrusion from the printing nozzle is controlled by a specific system using various interventions including pressure-based control, pneumatic or mechanical control, or solenoid control before forming layered printed ink as required by the computerized set up to build up the 3D biomaterial designs [7]. The biopolymer should have an excellent solid-to-melt transition property to produce high-resolution 3D cell-laden on the printer platform [31]. However, the extrusion-based 3D bioprinting potentially could generate high mechanical force

#### *Design and Manufacturing*

and shear stress together with high viscous of substrate lead to cellular apoptosis [5]. Further adjustment and optimization of this extrusion-based bioprinting can mitigate the drawbacks but it reduces the bioprinter resolution and speed [32]. Besides, the low concentration of ink viscosity supported cell proliferation and sustained the cell viability by introduced a composite-modified printing ink [33].

Light or laser-assisted 3D bioprinting, also known as stereolithography (SLA) (**Figure 3**), focuses on polymer resins manufacturing [19]. There are many variations of light or laser printing approaches for 3D fabrication. The advantages of these approaches are that they provide excellent accuracy, and good resolution between 10 and 50 μm [21]. This technique involves the patterning of a laser beam toward photo-based polymer to generate physical hardened polymer. This

#### **Figure 2.**

*The extrusion-based 3D bioprinting is easy to handle, customized-based design bioprinter, and versatile with the developed current system. It can be categorized into the fused deposition modeling and direct ink writing.*

#### **Figure 3.**

*Light- or laser-assisted 3D bioprinting approaches supported the high cell viability, accuracy, and good resolution between 10 and 50 μm. Two types consist of digital light processing-based bioprinting (DLP) and the two-photon polymerization-based bioprinting (TPP).*

**191**

*3D Printed Bioscaffolds for Developing Tissue-Engineered Constructs*

procedure is repeatedly applied to fabricate multi-layered polymer in the build-up stage. The other two types of laser-assisted 3D bioprinting techniques that are primarily applied in tissue engineering are digital light processing-based bioprinting (DLP) and the two-photon polymerization-based bioprinting (TPP) [34, 35]. The DLP technology uses a digitalized micro-mirror device chip (DMD) that contains around 2 million micro-mirrors. It functions to ensure light projection patterning precisely and is easy to modulate either on or off while the printing process is running on the platform. This technology consists of two 3D printing platform systems, namely, dynamic optical projection stereolithography (DOPS) and microscale continuous optical printing (μCOP) that support dynamic printing and continuous printing, respectively [7]. The TPP printing mechanism depends on the absorption of two photons by a molecule associated with light intensity square [36]. This phe-

nomenon contributed to the printing of voxel dimension below 1 μm3

printing approach is an ideal method to generate nanoscale and microscale printing 3D output. However, high-resolution printing limits the construct size and printing speed. Nonetheless, the TPP printing speed is still faster than that in extrusionbased bioprinting and at a similar rate with inkjet-based bioprinting [37].

Due to the limitation in technology to support the formation of the adequate and functional vascular network *in vitro*, currently, 3D bioprinting is more successful in the bioprinting of avascular tissue such as skin and cartilage. A complex tissue or organ with an extensive vascular network is still very challenging to prepare using the 3D bioprinting technology. To date, researchers are yet to succeed in preparing transplantable complex tissue or organ due to the difficulty in creating the circulatory system, especially the capillaries. However, several strategies have been used to improve the vascularization of 3D printed tissues, including printing of human umbilical vein endothelial cells (HUVECs) and vascular endothelial growth factors [38, 39] as well as seeding of endothelial cells and smooth muscle cells to the 3D

Bone tissue is one of the earliest tissues that were 3D printed and clinically used due to the ability of this technique to fabricate scaffolds according to the required shape, strength, and porosity. 3D printing enables fabrication of scaffold in any shape, which is not possible with many conventional fabrication techniques [41]. Furthermore, the materials commonly used for bone substitute production, such as hydroxylapatite (HA), synthetic calcium phosphate ceramics, polymethylmethacrylate, polylactides/polyglycolide and copolymer ceramics, tricalcium phosphate (TCP), bioglass, titanium, and other composite materials, are very compatible with the 3D printing technology [42]. The bone 3D printing had started as early as the 1990s, which utilized a powder-based freeform fabrication method [43]. Today, the bone substitute can be fabricated using the 3D plotting/direct ink writing, laser-assisted bioprinting (LAB), selective laser sintering (SLS), stereolithography (SLA), and fused deposition modeling (FDM) [42]. For example, Goriainov et al. prototyped hip joint implants using computer-aided design-computer-assisted manufacturing (CAD-CAM) and fabricated the scaffold using direct metal laser sintering from titanium alloy [44]. The custom-designed implants were seeded with autologous bone marrow aspirate before the implantation to 11 patients who

**3. 3D bioprinting for developing tissue substitutes for therapeutic** 

. Thus, this

*DOI: http://dx.doi.org/10.5772/intechopen.92418*

**applications**

printed tissues [40].

**3.1 Bone**

#### *3D Printed Bioscaffolds for Developing Tissue-Engineered Constructs DOI: http://dx.doi.org/10.5772/intechopen.92418*

procedure is repeatedly applied to fabricate multi-layered polymer in the build-up stage. The other two types of laser-assisted 3D bioprinting techniques that are primarily applied in tissue engineering are digital light processing-based bioprinting (DLP) and the two-photon polymerization-based bioprinting (TPP) [34, 35]. The DLP technology uses a digitalized micro-mirror device chip (DMD) that contains around 2 million micro-mirrors. It functions to ensure light projection patterning precisely and is easy to modulate either on or off while the printing process is running on the platform. This technology consists of two 3D printing platform systems, namely, dynamic optical projection stereolithography (DOPS) and microscale continuous optical printing (μCOP) that support dynamic printing and continuous printing, respectively [7]. The TPP printing mechanism depends on the absorption of two photons by a molecule associated with light intensity square [36]. This phenomenon contributed to the printing of voxel dimension below 1 μm3 . Thus, this printing approach is an ideal method to generate nanoscale and microscale printing 3D output. However, high-resolution printing limits the construct size and printing speed. Nonetheless, the TPP printing speed is still faster than that in extrusionbased bioprinting and at a similar rate with inkjet-based bioprinting [37].
