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

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 printed tissues [40].

#### **3.1 Bone**

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

were unsuitable for standard revision hip surgery. The postoperative results showed extensive new bone formation in the patients and a certain level of load-bearing function at the hip joint. The *in vitro* studies demonstrated the osteogenesis of the skeletal stem cells and osseointegration of the cells with the titanium alloy [44].

## **3.2 Skin**

The other tissue that has a high potential to utilize 3D printing technology to repair and regenerate is skin. Although skin substitutes made by conventional tissue engineering techniques such as Matriderm®, Integra®, Dermagraft®, and OrCel® have been commercialized and been used in clinics for wound treatment, there are still challenges that are yet to be resolved by these skin substitutes. These skin substitutes are expensive, require long production time with prolonged healing time, have limited tissue functionality, and resulted in scarring in some cases [45]. Besides, these skin substitutes lack hairs, sweat glands, sebaceous glands, and other skin appendages as well as pigmentation. The 3D bioprinting technology has led to the paradigm shift in the skin substitute production where this transformative technology enables simultaneous and accurate deposition of multiple types of skin cells, the formation of scaffolds with complex macro- and micro-architecture, creation of vascular networks, and construction of stratified layer [46].

The commonly used skin 3D bioprinting techniques are microextrusion, inkjet, stereolithography, and laser-assisted bioprinting [47]. The materials commonly used in skin 3D bioprinting are mainly natural polymers such as alginate, gelatine, collagen, fibrin, and hyaluronic acid. However, biocompatible synthetic materials such as polycaprolactone (PCL), polyglycolide (PGA), polyethylene glycol (PEG), poly(lactic-co-glycolic acid) (PLGA), and polylactide (PLA) are commonly combined with natural polymers to increase the mechanical strength of the skin substitute [46, 47]. The bio-inks serve either as the cell carrier or sacrificial support that is removed after the printing, or both as a carrier and mechanical support material that provides greater strength and microarchitecture that supports the function of the skin even after the implantation on to the patients [46]. The on-site bioprinting of either autologous or allogeneic dermal fibroblasts and epidermal keratinocytes directly into a wound area is the latest development in skin 3D bioprinting. The direct deposition of the cells in fibrinogen/collagen solution in a layer-by-layer method onto porcine full-thickness wound has shown to promote the wound closure, reduce contraction, and enhance the re-epithelialization, and the regenerated skin tissue had the composition similar to healthy skin [48].

### **3.3 Vasculature**

The other important and potential use of 3D bioprinting technology is the fabrication of vascularized tissues for passage of blood, air, lymph, and other vital fluids in the human body. The cells in dense tissue need to be within 200 mm from a vessel supplying oxygen and nutrients to survive [49]. The conventional technologies faced a major hindrance in fabricating vascular network structure in the dense engineered tissues, which is very crucial for the functioning of the implanted tissue or organ substitute, due to the technological limitation [50]. However, 3D bioprinting technology had enabled the fabrication of complex tissues with an integrated vasculature system, which in turn enabled the integration of the implant vasculature system with that of the host and long-term exchange of air, nutrient, and waste between the native and the implanted tissues [51].

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The construction of the vasculature network throughout the tissue is achieved through the design and fabrication of the hollow tube structure in the micrometer scale. This hollow structure is also seeded with vascular cell types or angiogenic factors to promote the formation of functional microvascular networks structure, especially the branching that can size-up to the nanoscale range and also permeation capability [51]. The two main additive manufacturing concepts used for vascularized tissue formation are indirect and direct printing. In the indirect printing, a sacrificial material or negative mold is printed using thermo-reversible hydrogels such as Pluronic F-127 in combination with another material as the permanent scaffold. Upon completion of the 3D printing, the sacrificial mold is removed to form the vascular network that was cellularized with vascular cells [52]. In the direct printing method, the vascular structure is actively printed either with cell-loaded biomaterial or cell-compatible bio-ink. The bio-ink utilized in this process normally has quick gelation/cross-linking ability, or extrinsically induced to crosslink/cured,

3D bioprinting has been utilized to prepare vascular networks in several studies. Miller et al. printed a 3D carbohydrate-glass lattice that was late embedded within an engineered tissue with living cells. Then, the 3D carbohydrate-glass lattice is removed, leaving interconnected hollow structures that can be seeded with endothelial cells to form the vasculature [53]. Later on, the same group of researchers proved that the vascular patch prepared using this technique can guide angiogenesis

Besides the tissues discussed above, various other tissues have been and being fabricated with the still-evolving 3D bioprinting techniques. Many of these 3D printed tissues had also been implanted on patients as part of a clinical study [55–57] and systematic clinical trials are also being conducted for many of these products, which have been reviewed by Mehrotra et al. [58]. The 3D printed implants are in the clinical trial phase mostly as implants for an ankle injury, bone fracture, disease and deformation, and breast reconstruction. Among the other tissues that are in lab-scale fabrication and optimization but have a high potential for therapeutic use are liver tissue [59, 60], cardiac tissue [61, 62], kidney tissue [63, 64], pancreas

Although the 3D bioprinting is a new technology, a few types of tissues produced by this technology are already utilized for therapeutic use. However, for the other tissues that have complex microarchitecture, and regulated by multiple signaling factors and cues from surrounding host tissues, it might need a longer time for the 3D printed tissue substitutes to be used in the clinical setting. The 3D printing of complex tissues needs more synergistic research from researches in various fields and various angles before it could fully mimic the native tissue's function. Another aspect to be considered will be the scaling up of the production using the clinicalgrade materials and commercial-scale 3D printers as most of the current studies are being done with experimental materials and lab-scale 3D printer technologies.

Personalized medicine, also known as precision medicine, is a concept in medicine that emphasizes that each patient should be managed differently based on an individual's condition. This tailored therapy shall be able to provide the

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

to maintain a stable hollow structure [52].

*in vivo* and rescue the ischemic tissues [54].

tissue [65, 66], cartilage [67, 68], and neural tissues [69, 70].

**4. 3D bioprinting for personalized therapy**

**3.4 Other tissues**

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

The construction of the vasculature network throughout the tissue is achieved through the design and fabrication of the hollow tube structure in the micrometer scale. This hollow structure is also seeded with vascular cell types or angiogenic factors to promote the formation of functional microvascular networks structure, especially the branching that can size-up to the nanoscale range and also permeation capability [51]. The two main additive manufacturing concepts used for vascularized tissue formation are indirect and direct printing. In the indirect printing, a sacrificial material or negative mold is printed using thermo-reversible hydrogels such as Pluronic F-127 in combination with another material as the permanent scaffold. Upon completion of the 3D printing, the sacrificial mold is removed to form the vascular network that was cellularized with vascular cells [52]. In the direct printing method, the vascular structure is actively printed either with cell-loaded biomaterial or cell-compatible bio-ink. The bio-ink utilized in this process normally has quick gelation/cross-linking ability, or extrinsically induced to crosslink/cured, to maintain a stable hollow structure [52].

3D bioprinting has been utilized to prepare vascular networks in several studies. Miller et al. printed a 3D carbohydrate-glass lattice that was late embedded within an engineered tissue with living cells. Then, the 3D carbohydrate-glass lattice is removed, leaving interconnected hollow structures that can be seeded with endothelial cells to form the vasculature [53]. Later on, the same group of researchers proved that the vascular patch prepared using this technique can guide angiogenesis *in vivo* and rescue the ischemic tissues [54].

#### **3.4 Other tissues**

Besides the tissues discussed above, various other tissues have been and being fabricated with the still-evolving 3D bioprinting techniques. Many of these 3D printed tissues had also been implanted on patients as part of a clinical study [55–57] and systematic clinical trials are also being conducted for many of these products, which have been reviewed by Mehrotra et al. [58]. The 3D printed implants are in the clinical trial phase mostly as implants for an ankle injury, bone fracture, disease and deformation, and breast reconstruction. Among the other tissues that are in lab-scale fabrication and optimization but have a high potential for therapeutic use are liver tissue [59, 60], cardiac tissue [61, 62], kidney tissue [63, 64], pancreas tissue [65, 66], cartilage [67, 68], and neural tissues [69, 70].

Although the 3D bioprinting is a new technology, a few types of tissues produced by this technology are already utilized for therapeutic use. However, for the other tissues that have complex microarchitecture, and regulated by multiple signaling factors and cues from surrounding host tissues, it might need a longer time for the 3D printed tissue substitutes to be used in the clinical setting. The 3D printing of complex tissues needs more synergistic research from researches in various fields and various angles before it could fully mimic the native tissue's function. Another aspect to be considered will be the scaling up of the production using the clinicalgrade materials and commercial-scale 3D printers as most of the current studies are being done with experimental materials and lab-scale 3D printer technologies.

#### **4. 3D bioprinting for personalized therapy**

Personalized medicine, also known as precision medicine, is a concept in medicine that emphasizes that each patient should be managed differently based on an individual's condition. This tailored therapy shall be able to provide the

best treatment plan for the patients to improve their prognosis. In personalized medicine, all the patient's specific characteristics such as age, gender environment, height, weight, diet, environment, and genetics are being considered during the prevention, diagnosis, and treatment phase. Personalized medicine can improve the quality of patient care and reduce the cost by avoiding unnecessary diagnostic testing and treatments [71–73]. Personalized medicine is not only limited to drugs but also for tissue engineering and regenerative medicine. Tissue engineering is highly personalized as a specific tissue-engineered substitute is needed for each patient. For example, different burn patients are presented with different degrees of injury and varied wound location, size, and dimension. Thus, a unique engineered skin needs to be prepared in the current good manufacturing practice (cGMP) facility for each patient.

3D bioprinting is one of the techniques that allow the preparation of personalized tissue-engineered substitutes. One of the major advantages of 3D bioprinting in the field of tissue engineering is the possibility of producing personalized living tissue comprising of stem cells, cell-friendly matrix, and bioactive compound in the dimension uniquely suited for different patients. 3D bioprinting can be used to print simple living tissues like skin to a more complex hollow structure like a trachea and very complex organ like heart and kidney. This is something other living tissue fabrication techniques cannot achieve as these techniques do not allow precise deposition of cells at the space wanted. With the advances in the 3D bioprinting technology, nowadays, it is possible to print multiple types of cells, biomaterials, and bioactive compounds at different spaces to create a complex tissue that mimics the native tissue cellular arrangement and mechanical properties. Maturation of the 3D printed tissues can be achieved using a bioreactor.

To prepare the personalized 3D bioprinted living tissue, the image of the targeted tissue in specific patients needs to be taken and reconstructed into 3D, which will be used to guide the 3D printer to print the tissue in the dimension wanted layer-by-layer to form the 3D tissue [74]. Initially, 3D bioprinting is used to prepare engineered tissue *in vitro*, which can be transplanted *in vivo* afterward. However, it is difficult to maintain the shape and size of the engineered tissue *in vitro*. Thus, researchers come out with the idea of 3D bioprinting the tissue *in situ*, directly on the defect site (**Figure 4**). *In situ* 3D bioprinting allows the precise fitting of the printed tissue to the defect site, which is unique for every patient. *In situ* 3D bioprinting might be more efficient compared to the conventional technique as it allows more accurate reconstruction of defect sites and harnesses the natural healing capacity of the body to mature the printed tissue on time. An *in situ* 3D bioprinters can be as simple as a portable handheld spray gun to a complex robotic arm-assisted 3D bioprinter. Di Bella et al. developed an *in situ* handheld 3D bioprinter that printed mesenchymal stem cells encapsulated within the hyaluronic acid methacrylate-gelatin methacrylamide hydrogel surrounded by the hyaluronic acid methacrylate-gelatin methacrylamide hydrogel + photoinitiator VA-086 shell, which can be photocured using the ultraviolet right for the treatment of cartilage defect [75]. Keriquel et al. used 3D bioprinted mesenchymal stem cells in collagen with hydroxyapatite for bone tissue engineering in a mice model [76]. Cohen et al. used a robot-assisted method of *in situ* 3D bioprinting for the deposition of alginate hydrogel and demineralized bone matrix-gelatin hydrogel for the regeneration of cartilage and bone defects, respectively [77].

Apart from personalized engineered tissue substitutes, 3D bioprinting also can be utilized for the preparation of personalized drug delivery systems and functional tissue models for personalized drug screening and disease modeling. Various models have been developed, including the liver [78], heart [79], blood vessel [80], skin [81],

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

skeletal muscle [82], and cancer [83]. The development of these models can greatly improve the medical care the patients will receive as distinctive prevention and treat-

The invention of 3D bioprinting has revolutionized biomedical research and significant development in translational research closing the gap from bench to bedside. In the pharmaceutical industry, the value of 3D bioprinting is expected in lowering the attrition rate of a new drug since 3D bioprinting has the potential to precisely position multiple cell types as needed according to the tissue of interest (**Figure 5**). Thus, 3D bioprinting enables a more robust design of drug screening, drug delivery, high-throughput drug testing, and ADME assays. The application of 3D bioprinting in the development of in vitro tissue or organoid models for drug

The ability of 3D bioprinting in replicating tumor microenvironment (TME) provides a better model to assess drug response, tumor proliferation, and metastasis. By 3D bioprinting, a tumor model with hypoxic core and necrosis could be recreated similar to the *in vivo* environment [84, 85]. The 3D-printed glioma model comprising of glioma stem cells incorporated in alginate/gelatin/fibrinogen bioink is an example, and it showed higher resistance to temozolomide than in a 2D culture model [86]. Another case in point, fabrication of breast cancer model was achieved via the Organovo 3D NoveGen Bioprinter system where cancer cells are bordered with a stromal milieu of endothelial cells, fibroblast, and adipocytes. The said breast cancer model was viable for up to 14 days and possesses distinct internal compartmentalization. The model has been used to test hormonal drug response

**5. 3D bioprinting for developing in vitro tissue/organoid models** 

*The personalized 3D bioprinted living tissue has been printed layer-by-layer to form the 3D tissue.*

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

ment strategies can be designed individually.

**for drug discovery**

**Figure 4.**

discovery is discussed in this section.

**5.1 Tumor or cancer model**

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

**Figure 4.** *The personalized 3D bioprinted living tissue has been printed layer-by-layer to form the 3D tissue.*

skeletal muscle [82], and cancer [83]. The development of these models can greatly improve the medical care the patients will receive as distinctive prevention and treatment strategies can be designed individually.
