*2.3. Biomorphic Transformations*

A valuable approach to obtain bioceramic composite scaffolds with a complex structural hierarchy relies on biomorphic transformations of natural structures mimicking the morphology and microstructure of the target tissue [20,77,79].

Since the 1970s, biomorphic transformations from natural sources have been proposed for the fabrication of bioceramic scaffolds due to their 3D highly interconnected porous architecture, including the replica of the porous microstructure of CaCO3-based corals, which are impossible to create artificially, and the replica of marine sponges, soft vegetal structures and fruit- and wood-template bioceramics [75,80,81].

The approach of wood biotransformation is particularly interesting, as many ligneous species exhibit a porous and hierarchically organized structure very close to that of cortical and cancellous bone. The transformation of wood generally involves pyrolysis followed by a hydrothermal treatment; in particular, a complex multi-step strategy to convert rattan wood structures into biomimetic HA scaffolds was proposed [76,77,82]. In particular, several subsequent and strictly controlled reactions are required, including (i) the *pyrolysis* of wood to produce a carbon template; (ii) *carburization*, calcium infiltration to transform carbon in CaC2; (iii) an *oxidation* process that leads to CaO formation; (iv) *carbonation* by hydrothermal processes or by heterogeneous processes carried out at supercritical conditions and high pressure; and finally (v) *phosphorylation* through the hydrothermal process generating biomimetic, hierarchically organized scaffolds made of ion-doped HA.

#### *2.4. 3D Printing*

Three-dimensional (3D) printing represents an additive manufacturing (AM) technique (also known as rapid prototyping) to produce complex-shaped devices with complex geometry and design flexibility from 3D model schemes [83–86]. A wide range of materials have been employed with 3D printing techniques, including metals, polymers, ceramics and composites [85,86].

Different 3D printing methods have been proposed [85,87–90]. Extrusion-based techniques consist of the deposition of ink to create designed structures by forcing the ink through a nozzle as a melt, in fused deposition modeling (FDM), or viscous suspensions, in direct ink writing (DIW), to form lines that solidify onto a build plate [90].

DIW represents an easy manufacturing technique that allows the creation of a wide range of structures, from solid monolithic parts to highly complex porous scaffolds and composite materials. The use of pastes also allows shape retention due to the high solid loading and visco-elastic properties. The use of high viscous inks requires larger diameter nozzles compared to the conventional inkjet printing ink; it can therefore be used successfully to print extremely viscous pastes that are HA based [88].

Three-dimensional printing technology finds a wide range of biomedical applications: craniofacial implants, dental models, prosthetic parts, scaffold for tissue regenerations (bone and skin), organ printing, tumor therapy and tissue modeling for drug discovery [90–92]. In these kinds of applications, printable materials are formulated from biomaterials and bioinspired materials to achieve patient-specific scaffolds with high structural complexity [93]. Moreover, printable biomaterials should be biocompatible and bioactive and should have good degradation kinetics, appropriate mechanical properties, give desirable cellular responses and exhibit tissue biomimicry [94,95].

Bioceramic powders, natural or synthetic hydrogels, polymers and their composites have been used as raw materials to formulate inks for 3D printing; in this review, we focused on ceramic-based scaffolds and bioceramic/polymer composites. Bioceramics commonly printed are calcium phosphate-based bioceramics (HA, TCP and biphasic CaP), calcium silicate-based bioceramics and bioactive glasses [91,93].

Moreover, the precise tuning of the macro- and micro-porosity permitted by 3D printing technology not only allows the fabrication of scaffolds with hierarchical porosity but also leads to the controlled release of biomolecules or drug loaded in the scaffold matrix or adsorbed on the scaffold surface [96,97].

Three-dimensional-printed bioceramics include sintered 3D-printed bioceramics, non*sintered 3D-printed bioceramics* and *composites with polymers*.In the first case, bioceramic scaffolds are printed and sintered, removing the organic phases and improving the mechanical properties of the structure [93]. In the presence of biologically active ions, such as magnesium or strontium, in addition to an improvement of mechanical properties, an increase in biological performance in vivo was also reported [98]. Another study described biphasic CaP scaffolds (HA:β-TCP with a weight ratio 60:40) coated with calcium peroxide and polycaprolactone in order to promote bone growth with greater proliferation of osteoblasts under hypoxic conditions, following the release of oxygen dependent on the concentration of calcium peroxide in the PCL coating [99].

In non-sintered 3D-printed bioceramics, a small amount of organic solvent is used as a binder for bioceramic powders and is not removed after printing. Sun et al. developed a porous 3D scaffold of biodegradable CaP loaded with antibiotics for the regeneration of the bone tissue of the jaw, achieving a controlled drug release. This scaffold was based on an HA or biphasic mixture of CaP (β-TCP and HA with a weight ratio of 1:1) cross-linked with sodium alginate in the presence of the drug, and the paste was then extruded by the 3D printer. By modulating the degree and the time of cross-linking, it is possible to control the drug release kinetics. In vitro studies show low cytotoxicity and good cell adhesion and proliferation on the scaffold surface [100].

Bioceramic and polymer composite are synthesized to combine the bioactivity and osteoconductivity of bioceramics with the handling performance of polymers [87]. For example, the presence of strontium-doped HA nanoparticles in 3D-printed PCL scaffolds leads to a significant increase in cell proliferation and bone regeneration, due to the simultaneous release of calcium and strontium ions, associated with an improvement in mechanical properties as related to the inorganic phase content [101]. HA nanoparticles were also used as an external coating for 3D-printed polymer scaffolds in order to enhance cell proliferation and differentiation while also strengthening the scaffold [94].

Recently, 4D printing approaches have been developed, which, in addition to threedimensional spatial control, introduces the concept of temporal control, i.e., active smart materials responsive and mechanically converted into other shapes via external stimuli. This technique enables the production of smart 3D scaffolds responding to external stimuli, such as changes in pH and temperature or when subjected to magnetism or light radiation of adequate energy [95,102,103].

#### **3. Enhancing the Biological Performance of Bioceramic Composites**
