# Bone Substitutes Scaffold in Human Bone: Comparative Evaluation by 3D Micro-CT Technique
## 4 Discussion  
The structural characterization of the biomaterials is a relevant step for the investigation of the bone substitute properties before implantation and it should be applied to choose the most suitable biomaterial for a specific clinical application. The porosity is a well-known relevant aspect for scaffolds in bone tissue engineering applications [2,4,8]. A high porosity improves the invasion of the graft by progenitor cells. The higher the percentage of pores, the greater the migration of mesenchymal cells and osteoblasts to the recipient site. In addition, a high porosity and a high contact surface-volume ratio, i.e., the presence of many and small pores, promote cell adhesion. At the same time, large pores and the presence of large interconnections among them, improve osteogenesis allowing adequate vascularization. Therefore, an ideal scaffold should have features such as high porosity, a large surface area available for cell adhesion, sufficiently large pores and extensive interconnectivity allowing the penetration of newly formed vascular structures. In the stages following implantation, complex processes of cell migration, differentiation and proliferation occur, which ultimately lead to bone neformation. The presence of a 3D osteoconductive matrix that has structural characteristics as similar as possible to human bone tissue greatly facilitates the regenerative process [16]. Some authors [4, 5, 6] have shown that pores between 100 and 300 \(\upmu\)m are needed to improve osteogenesis, but large pores could compromise the mechanical strength. This last aspect is relevant at the initial steps of the process as the graft should be able to act like a support of the skeletal segment. From the point of view of the mechanical characteristics of the graft, the size and thickness of the trabeculae are also relevant. Thus, it is necessary to establish a compromise for an optimal performance of the graft on the recipient site. The iliac crest is the most commonly used extra-oral donor site of autologous bone in the various techniques of bone regeneration [34]. In this study, it was only addressed as a reference at the preclinical phase, before performing the clinical study and without the intention to select the best biomaterial. Preclinical results obtained from the microtomographic analyses (Table 2), showed that the bone substitutes with greater similarities with the human counterpart are the biomaterials of bovine origin-1a and equine-1b. Sufficient porosity and adequate values of BS/BV and Tb.Sp allowed and facilitated entry and migration within the newly formed bone tissue scaffold. At the same time, the BV/TV and the Tb.Th guaranteed mechanical properties comparable to the human bone tissue. The values  
Figure 6: Histology images of specimens test (left side) and control site (right side): (**A1**) 1a test, entire specimen; (**A2**) 1a control; (**B1**) 2b test; (**B2**) 2b control; (**C1**) 4a test and (**C2**) 4a control; Legend: CT = Connective Tissue; Sc = residual Scaffold; NB = New Bone; BV = Blood Vessels. obtained for the two synthetic derivatives, calcium triphosphate, and polylactic-polyglycolic acid, i.e., 3a and 4a, evidenced and, confirmed for the 3a, that these biomaterials had greater compactness compared to the previous one--2b. In particular, the 4a sample was characterized by a lower porosity and Tb.Sp, and higher BV/TV; consequently, less space was available for colonization by osteogenic cells and newly formed vessels. Higher interconnection among the internal empty spaces, evidenced in the 3D reconstructed images, is fundamental to be considered for the migration of cells and fluids in the depth of a scaffold; the lower porosity and greater values of the BV/TV and the Tb.Th suggest greater mechanical resistance of these scaffolds. Regarding the results of both parts of the clinical study, in all the examined cases, the specimens of the test site showed a minor compactness with a higher volume of non-mineralized tissue.