*3.1. Biofunctionalization*

Biofunctionalization is the modification of a material to achieve improved biological function and/or stimulus, whether permanent or temporary. The biofunctionality of scaffolds for regenerative medicine has been considered to play a key role for effective tissue regeneration [92,95].

Several parameters can be tuned, including surface energy and roughness, Ca/P ratio, solubility, particle size and crystallinity, in order to improve the biological events beyond the interaction with the biological environment, e.g., protein adsorption, cell attachment, cell proliferation and cell differentiation [93,104] (Figure 2).

**Figure 2.** Key properties of CaP-based bioceramics that have an impact on biological events.

The architecture of biomimetic scaffolds greatly affects the chance to obtain a suitable microenvironment for bone regeneration. The presence of a diffuse macroporosity favors cell adhesion, cell proliferation and vascular growth. In turn, the surface micro-architecture enhances protein adsorption, and specific nano-topography could directly modulate the osteogenic differentiation, producing a favorable osteoimmune microenvironment [97]. Among the various microstructures, microgrooves have strong effects in the regulation of cell orientation and adhesion [96,105]. The width of the micro-channels controls the orientation, while the depth regulates the adhesion strength of the cells, which decreases as the depth of the groove increases. Micro–nano hybrid structures (micropattern–nanorod hybrid structure) showed higher cell adhesion, proliferation and ALP (alkaline phosphatase protein) activity than a single-scale structure (including nanorods and micropatterns) [96,106].

The roughness of the surface plays a crucial role in cellular behavior [107–109] (Table 8).

**Table 8.** Effects of structural size, morphology and roughness surface of CaP biomaterials on cellular behavior.


It was demonstrated that specifically designed roughness can enhance osteogenesis due to the modulated concentration of calcium ions and osteocalcin in the grooves [110].

The surface chemistry also plays a key role in cell behavior. The crystallinity of nanometric bioceramics, i.e., ACP and HA, was observed to affect cell attachment efficiency, proliferation and differentiation of bone marrow-derived mesenchymal stem/stromal cells (BMSCs) [111]. In particular, nano-HA allows a better adhesion, proliferation and differentiation of BMSCs into osteoblasts than ACP.

The chemical approach of creating functional groups on the surface of the scaffolds is also promising for the improvement of cell adhesion and osteogenic differentiation. For example, functional groups, such as –COOH and –NH2, improve protein adsorption due to the formation of hydrogen bonds linking proteins, finally resulting in improved cell adhesion [97].
