**Engineered Tissue**

**Figure 1.** Vital elements of tissue engineering (simplified diagrammatic representation of the basic **Figure 1.** Vital elements of tissue engineering (simplified diagrammatic representation of the basic concept of tissue engineering, i.e., scaffold, cells, and growth-stimulating factors are the three essential parameters responsible in tissue engineering for forming new functional tissue).

concept of tissue engineering, i.e., scaffold, cells, and growth-stimulating factors are the three essential parameters responsible in tissue engineering for forming new functional tissue). The bioreactor uses this triad to imitate a natural environment to reproduce and grow new functional tissues or cellular components. Figure 2 shows an illustration of the basic principle of TE.

The bioreactor uses this triad to imitate a natural environment to reproduce and grow new functional tissues or cellular components. Figure 2 shows an illustration of the basic principle of TE. Firstly, cells are isolated from a biopsy (allogenic, syngeneic, xenogeneic, or autologous source) and allowed to grow and expand in vitro, in a cell culture system, or in a bioreactor. The expanded cells are then seeded onto a nutrient and growth factors-rich matrix or carrier (scaffold) for structural support. Here, the cells grow, differentiate, and proliferate to form new tissues, then migrate to the carrier to replace the old tissues. Lastly, this TE product will be grafted into the patient to replace the damaged tissues [11].

**Figure 2.** An illustration of the basic principle of TE, which includes cell isolation, cell culture, cell **Figure 2.** An illustration of the basic principle of TE, which includes cell isolation, cell culture, cell expansion, and tissue grafting into the patient's body.

#### *2.1. Key Elements of Tissue Engineering*

expansion, and tissue grafting into the patient's body.

#### Firstly, cells are isolated from a biopsy (allogenic, syngeneic, xenogeneic, or autol-2.1.1. Cells

ogous source) and allowed to grow and expand in vitro, in a cell culture system, or in a bioreactor. The expanded cells are then seeded onto a nutrient and growth factors-rich matrix or carrier (scaffold) for structural support. Here, the cells grow, differentiate, and proliferate to form new tissues, then migrate to the carrier to replace the old tissues. Lastly, this TE product will be grafted into the patient to replace the damaged tissues [11]. *2.1. Key Elements of Tissue Engineering*  2.1.1. Cells The cell is a structural and functional unit of life in all living organisms. Cells performing the same function are grouped to form tissues and create a body system. While designing a TE product, especially for clinical applications, cell source selection becomes a crucial issue, as it determines the success of the tissue generation step. Essentially, the cells isolated for TE applications should fulfill the essential requirement of combining themselves with the selected tissue with different growth factors and cytokines that activate the endogenous tissue regeneration program. However, natural cells have difficulty reproducing the same particular cell type in large quantities. A promising cell source called stem cells is then developed as an alternative. Stem cells can be categorized into embryonic (ESCs), adult (ASCs), and induced pluripotent stem cells (iPSCs). ESCs The cell is a structural and functional unit of life in all living organisms. Cells performing the same function are grouped to form tissues and create a body system. While designing a TE product, especially for clinical applications, cell source selection becomes a crucial issue, as it determines the success of the tissue generation step. Essentially, the cells isolated for TE applications should fulfill the essential requirement of combining themselves with the selected tissue with different growth factors and cytokines that activate the endogenous tissue regeneration program. However, natural cells have difficulty reproducing the same particular cell type in large quantities. A promising cell source called stem cells is then developed as an alternative. Stem cells can be categorized into embryonic (ESCs), adult (ASCs), and induced pluripotent stem cells (iPSCs). ESCs are pluripotent cells that can differentiate into any desired lineage, but are ethically controversial and have a shortage in teratoma production [12]. ASCs are multipotent cells and are considered more appropriate for TE applications than ESCs. Though ASCs have more limitations in cell differentiation, they are believed to be less prone to rejections after transplantations. Therefore, ASCs are commonly used to isolate tissues such as bone marrow, muscle, adipose tissue, and umbilical cord [13]. iPSCs, on the other hand, are somatic cells in the pluripotent state that exhibit autologous characteristics and fulfill differentiation capacity [12]. Nevertheless, iPSCs are yet to be extensively used due to the need for precise characterizations of reprogramming the somatic cells before clinical applications [14].

are pluripotent cells that can differentiate into any desired lineage, but are ethically controversial and have a shortage in teratoma production [12]. ASCs are multipotent cells

#### 2.1.2. Growth Factors

Growth-stimulating signals include growth factors (GFs), which are a heterogeneous group of polypeptides bonded to specific receptors on the cell surface that regulates a heterogeneous group of polypeptides bonded to specific receptors on the cell surface that regulate cellular responses such as cell proliferation and cell survival, as well as the growth of targeted tissues [15]. Some GFs that have been used in TE applications include bone morphogenetic proteins, vascular epithelial growth factor (VEGF), and transforming growth factor-β (TGF-β) [16].

#### 2.1.3. Scaffolds

Scaffolds play an important role in TE applications, serving as a temporary platform or template for providing guidance and structural support to develop new tissues [17]. Scaffolds refer to a three-dimensional (3D) porous biomaterial that provides a favorable environment for cells to repair and regenerate tissues and organs [3]. It serves as a template for tissue defect reconstruction while promoting cell attachment, proliferation, extracellular matrix regeneration, and restoration of nerves, muscles, and bones. In addition, scaffolds can transport bioactive materials such as drugs, inhibitors, and cytokines as a mechanical barrier against the infiltrating native tissues, which may disturb tissue restoration and regeneration [11].

#### *2.2. Requirements of Scaffold*

## 2.2.1. Microarchitecture

The microarchitecture of the scaffold includes the porosity and pore size and the interconnectivity between the pores. Firstly, the pore size must be adequate for cell migration and attachment onto scaffolds. This also ensures proper mass transfer of nutrients and waste materials into and out of the cells and tissue or vascularization and infiltration. As suggested by Peri´c Kaˇcarevi´c et al., a smaller pore size is favorable, between 75 and 100 µm in vitro, while the maximum pore size should lie between 200 and 500 µm in vivo to allow optimal tissue penetration and vascularization [18,19]. Moreover, an interconnecting porous system is required to provide a larger scaffold surface area for cell attachment. In addition, having a higher porosity helps to maximize cell-to-cell interactions, thereby promoting the integration of the engineered tissues with the native tissues [20]. Alonzo et al. suggested a pore network comprising more than 60 percent of pores with pore diameters ranging between 150 and 400 µm and at least 20 percent smaller than 20 µm [21].

#### 2.2.2. Biodegradability

As scaffolds only act as a temporary platform for developing cells or tissues, they should be chemically or enzymatically broken down over time when grafted into living organisms. The rate at which the scaffold materials are broken down is known as biodegradability [3]. Ideally, the biodegradation rate of the scaffold should be proportional to the rate of new bone formation or tissue regeneration. When new tissues are successfully engineered and integrated with host bone, they will replace the biomaterial scaffolds via a "creeping substitution" step [22]. The non-toxic products of the scaffold will then be recycled as metabolites in other biochemical reactions or exit the body without interference with other organs and surrounding tissues [18,20].

#### 2.2.3. Biocompatibility

Furthermore, the scaffold should be highly biocompatible for cell adhesion and proliferation. There should be negligible chronic immune responses to prevent severe inflammatory reactions that might affect healing or cause rejection in the body. Even when inflammatory reactions occur, they should be recovered in no more than two weeks [23,24].

#### 2.2.4. Bioactivity

Scaffold bioactivity refers to its ability to interact with the surrounding cellular components of the engineered tissues. Unlike traditional passive biomaterials, which generally pose low or no interactions with the environment, bioactive scaffolds are designed to enhance proper cell migration or differentiation, tissue regeneration or neoformation, and integration in the host, thereby avoiding processes such as scarring [19]. Moreover, the scaffolds may be attached to cell-adhesive ligands to promote cell attachment, or to physical indicators such as topography to enhance cell morphology and alignment. In addition, bioactive scaffolds may serve as a transporter or reservoir for growth-stimulating signals such as GFs to enhance tissue regeneration [7].

#### 2.2.5. Mechanical Properties

Furthermore, the scaffold materials should pose similar intrinsic mechanical properties as native bones or tissues in the anatomical site of implantation. The mechanical properties of tissue vary in nature, as listed in Table 1. It provides structural support and shape stability and, at the same time, helps to minimize the risk of stress shielding, implantrelated osteopenia, and subsequent re-fracture. Moreover, the scaffold should also be strong enough to allow surgical handling during transplantations. Some examples of mechanical properties include elastic modulus, tensile strength, fracture toughness, fatigue, and elongation percentage [7,18,19,25].

**Table 1.** Young's modulus of various tissues.


Tensile testing and compressive testing are the conventional methods used to characterize the mechanical properties of a scaffold. Compressive/tensile strength, toughness, and Young's modulus are the important obtained parameters. No limitations for the geometrical structure of the specimen is the biggest advantage of compressive testing over tensile testing. Atomic force microscopy (AFM), dynamic mechanical analysis (DMA), rheometry, and micro indentation are the alternative methods for the characterization of mechanical properties [25]. Elasticity (Young's modulus), shear strength, and viscoelasticity measurement are some significant mechanical properties in cardiac tissue engineering. Due to its thin geometric structure (µm thickness), it is inadequate for DMA. Hence, viscoelasticity measurement for the cardiac scaffold is incorporated only in a few studies [35]. For the healing process, to endure osteogenic loads, adequate compressive strength is needed in bone tissue engineering [36]. Compared to other tissues, neural tissues have low mechanical stiffness with the range of 0.1 KPa for Young's modulus [37]. Mechanical properties play a crucial role in skin tissue engineering to resist physiological forces such as nerve bundles, vascular networks, and collagen deposition during the wound healing process [38]. Figure 3 depicts the requirements to be considered while developing the scaffold.

#### **Biocompatibility**

Hence, viscoelasticity measurement for the cardiac scaffold is incorporated only in a few studies [35]. For the healing process, to endure osteogenic loads, adequate compressive strength is needed in bone tissue engineering [36]. Compared to other tissues, neural tissues have low mechanical stiffness with the range of 0.1KPa for Young's modulus [37]. Mechanical properties play a crucial role in skin tissue engineering to resist physiological forces such as nerve bundles, vascular networks, and collagen deposition during the wound healing process [38]. Figure 3 depicts the requirements to be considered while

> (Cells must adhere, function & withstand against immune reaction)

**Manufacturing Technology**

developing the scaffold.

(Easy scale up and cost effective)
