*6.1.1 Methods*

In the first years of the emergence of 3D printing, conventional methods of this technique such as fused deposition modeling (FDM), stereolithography (SLA), and selective laser sintering (SLS) were used to print tissue engineering structures. Although these methods provided unique capabilities compared to the traditional scaffold construction methods, however, as time passed and the disadvantages of these methods became clear, such as the inability to load cells and bioactive molecules, the inadequate resolution, and the impossibility of using a wide range of materials led researchers to develop 3D bio-printing technology. In recent years, the use of 3D bio-printing technology has accelerated. This method is divided into three main types: inkjet-based bioprinting, extrusion-based bioprinting, and laser-assisted bioprinting [136–139]. Inkjet-based bioprinting includes the deposition of bio-ink droplets onto a substrate. Although this method can print a cell-laden scaffold at a high speed, the number of biomaterials that can be used in an inkjet printer is limited, due to the rapid gelation time requirement. The extrusion-based bioprinting method is extruding high cell density laden bio-inks to create scaffold structures. It is the most easiest and popular 3D bio-printing method. In laser-assisted bioprinting, the bioinks are printed using the laser in a nozzle-free manner with higher resolution. The laser heats the determined points and deposits the cells in the exact place [140].

### *6.1.2 Bone model optimizing*

As mentioned before, the 3D bio-printing can create personalized complex structures. The computer modeling of the 3D bio-printing structures has two main steps. The first step is data acquisition using computed tomography (CT) or magnetic resonance imaging (MRI) and the second step is image processing and model generation [141]. The 3D models should be predictable and shows controllable shape change. Therefore, the 3D model design should be optimized by considering chemical composition, volume fraction, and structural features such as pore size, pore shape, pore distributions, and mechanical properties [142]. The finite element analysis can be used to calculate the mechanical properties of the designed structures and predict the results, theoretically. Liang et al. [143] demonstrated the tetragonal structures showed better mechanics than the tetragonal, hexagonal, and wheel-like designed structures for bone replacements using finite element analysis and an electromechanical universal testing system. The combination of state-of-the-art optimization

techniques with computer models can provide a basis for predicting scaffold behavior and optimizing the designed scaffolds for bone regeneration, especially for large bone defect regeneration [144].
