**6. Scaffolds fabrication methods**

Scaffolds act as a temporal template for cell attachments and growth that can contain different cell types, growth factors, drugs, and bioactive molecules. Although the material selection had a great impact on the scaffolds' properties, the scaffold's fabrication technique is also an important criterion. There are different techniques for scaffold fabrication such as gas-foaming, phase separation, particle leaching, electrospinning, freeze-drying, freeze-casting, and 3D bio-printing. An ideal scaffold should be able to create the most similar structure to the natural bone tissue along the same physical, chemical, and biological properties while providing reproducibility with minimal cost and time. The conventional scaffold fabrication methods showed poor controllability during the creation process to fabricate complex structures with tunable micro-scale and macro-scale. The recent scaffold fabrication methods such as 3D bio-printing allow high controllability, the capability of complex structures, and the inclusion of the cells and growth factors in the structure [128–130].

#### **6.1 Three dimensional bio-printing**

Recently, 3D bio-printing is introduced as a novel scaffold fabrication method and has provided numerous capabilities compared to the traditional methods such as a lower rate of residual organic solvents which decreases the harmful effects of these chemical solvents on the cell's survival and interactions, or scaffold designing before printing which leads to personalization [131, 132]. Also, it is possible to build complex structures with/without cells [133]. The most important benefit of 3D bio-printing for bone tissue engineering is controlling cell distribution [134]. The printing materials that contain cells are called 'bio-ink' and should demonstrate some features for cell survival and the creation of a favorable bone substitute. The most important features in the bio-inks selection are biocompatibility, printability, fidelity, viscoelasticity, shear-thinning, yield stress, shelf life, cross-linking capability, and even cost [135].
