## 1 Introduction  
The regeneration of a three-dimensional (3D) tissue is favored by the use of scaffolds, 3D structures that support the cellular architecture. 3D cell cultures have developed rapidly in recent years as they have advantages over 2D cultures, being able to mimic the natural 3D organization of cells and their extracellular matrix (ECM). Therefore, since it has been proven that cell differentiation, migration, morphogenesis, and proliferation are influenced by its biochemical microenvironments, cell-cell and cell-ECM interactions that occur in 3D cell cultures are as representative as in vivo. Moreover, many desired cellular characteristics are maintained or even promoted in 3D cultures, supporting their use in basic and translational research [1]. Biodegradable scaffolds play an important role in creating a 3D environment to induce tissue formation and, together with stem cell technologies, are believed to hold enormous potential for tissue regeneration. Different biomaterials, natural and synthetic, bioresorbable and permanent, have been studied for their production. Silk fibroin (SF), a fibrous protein produced by silkworms _Bombyx mori_[2,3], thanks to its extraordinary mechanical, physical, biological, and biodegradable properties, gained great attention for biomedical applications, and it is considered a promising biomaterial [4,5] for bone tissue engineering [6] used to generate scaffolds in various formats such as hydrogels, spheres, capsules, films, and sponges [7, 8, 9, 10, 11]. The design and the characteristics of the scaffolds are relevant for the achievement of their role, the interaction with tissues, and the final efficacy [12,13]. In searching for the best reasonable solution [14], many studies have been conducted considering various compositions and multilayer scaffolds [15] and underlining the relevance to control the structures at various levels. In general, a good scaffold should be optimized in achieving its role in order to guarantee mechanical properties and a morphology able to allow communication inside the structure, preserving the strength. Various techniques and methodologies are often applied for a complete assessment before and after the interaction with tissues. Micro-computed tomography (micro-CT) is an established non-destructive technique to investigate biomaterials, bone tissue, scaffolds, and devices for applications in dentistry, maxillofacial surgery, tissue engineering, and regenerative medicine [16, 17, 18, 19]. It is an X-ray based medical imaging analysis that was very useful for the volumetric analysis of the structural and morphological properties, to detect accuracy and defects of the complex manufacturing process, and to quantify porosity, surface area, pore distribution and structural thickness, with high-resolution at a micron level [20, 21, 22, 23]. The micro-CT was demonstrated to be effective in enhancing the investigations if used in an integrated approach with other more traditional analyses and with a great potential if associated with other innovative techniques [17]. The most common way to cultivate cells in tissue engineering is to use a scaffold on which specific cells can be seeded [24]. Recently, there has been a growing focus on potential applications of mesenchymal stromal/stem cells (MSCs) [25]. MSCs play an important role in tissue regeneration and multi-lineage differentiation because of their intrinsic properties of self-renewal, pluripotency in mesodermal and non-mesodermal lines, and strong secretory activity of biological molecules and growth factors [26]. MSCs can be isolated from a wide variety of adult tissues (bone marrow, dental pulp, and adipose tissue) and fetal umbilical cord tissue. The MSCs from human Wharton's jelly of the umbilical cord (WJ-MSC) are easier to isolate and expand than MSCs from other fetal and adult tissues [27, 28, 29, 30]. Although WJ-MSC are generally considered a more primitive cellular population, they share many properties with their adult bone marrow counterparts [31].